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Effect of topical N-acetylcysteine in the prevention of postoperative pericardial adhesion formation in a rabbit model
Cardiovascular Pathology 22 (2013) 368–372
Contents lists available at ScienceDirect
Cardiovascular Pathology
Orginal Article
Effect of topical N-acetylcysteine in the prevention of postoperative pericardial adhesion formation in a rabbit model Necmettin Colak a,⁎, Yunus Nazli a, Mehmet Fatih Alpay a, Omer Nuri Aksoy a, Ismail Olgun Akkaya a, Reyhan Bayrak b, Omer Cakir a a b
Department of Cardiovascular Surgery, University of Fatih, Faculty of Medicine, Ankara, Turkey Department of Pathology, University of Fatih, Faculty of Medicine, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 3 July 2012 Received in revised form 21 January 2013 Accepted 6 February 2013 Keywords: Cardiac operation N-acetylcysteine Pericardial adhesion
a b s t r a c t Background: N-acetylcysteine (NAC), a precursor of reduced glutathione, has been in clinical use primarily as a mucolytic. In addition, NAC is well known for their free radical scavenging and antioxidant properties. Increasing of reactive oxygen products occurring during cardiac surgery can play an important role in postoperative adhesion formation. We investigated to the efficacy of the NAC for postoperative pericardial adhesions. Methods: Sixteen New Zealand white rabbits (2.5–3 kg) were used and categorized into two groups including study (use of NAC) and control groups. In both groups, the pericardium was opened longitudinally, and the exposed epicardial surfaces were abraded with dry gauze. The rabbits were divided into two groups: Group 1 was treated with the sponge, which impregnated with NAC solution, (10%, 300 mg/3 ml) and applied over the abraded epicardium for 5 min (n=8). Group 2 was the control, and the sponge, which was impregnated with 3-ml isotonic NaCl solution (0.9%), was applied onto the surface of the abraded epicardium for 5 min (n=8). After a period of 2 weeks, the animals were sacrificed. The scores of adhesion were graded by macroscopic examination, and the pericardial tissues were analyzed microscopically in point of inflammation and fibrosis. Results: In Group 1, the adhesion scores were significantly lower compared with the control group [Group 1 vs. 2; 1 (1–2) vs. 3 (2–3), Pb.001]. No significant difference was found between the groups in terms of the severity of inflammation [Group 1 vs. 2; 1.5 (1–3) vs. 2.5 (1–3), P=.083]. There was a difference between groups in terms of the degree of fibrosis [Group 1 vs. 2; 2 (1–2) vs. 3 (2–3), P=.007]. Conclusions: The use of NAC for preventing postoperative pericardial adhesions was reduced to adhesion and fibrosis scores in an experimental rabbit model. There was no statistically significant difference between groups in terms of inflammatory scores. The NAC effectively prevented the formation of pericardial adhesion. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Cardiac surgical procedures routinely results in the development of extensive fibrous adhesions. The condition may be general or localized and may involve adhesion between the epicardium and the pericardium, sternum, pleura, or surrounding tissues such as the diaphragm or other mediastinal structures, as a result of an inflammatory process. The presence of these adhesions not only adds to the operating time but also increases the risk of damage to the heart, great vessels, and previously placed grafts [1]. Especially, retrosternal adhesions resulting from previous surgery continue to be a challenge during resternotomy. Different techniques were applied to reduce or prevent development of postoperative adhesions in the The financial support from an institution was not taken in this study. ⁎ Corresponding author. Alparslan Turkes caddesi No:57, 06510, Emek/Ankara, Turkey. Tel.: +90 3122035187; fax: +90 3122213670. E-mail address: [email protected] (N. Colak). 1054-8807/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.carpath.2013.02.001
retrosternal spaces and mediastinal structures. Fibrinolytic agents, anticoagulants, antiinflammatory agents, several natural and synthetic physical barriers, and antibiotics have been tried with variable success [2–4]. Despite continuous advances and research, an ideal method to prevent postoperative pericardial adhesion formation has not been confirmed. N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine, is an excellent source of sulfhydryl (SH) groups and is converted in the body into metabolites capable of stimulating glutathione (GSH) synthesis, promoting detoxification, and acting directly as free radical scavengers [5]. The antioxidant activities of NAC have been reported in previous studies [6]. In addition, another study was reported varying doses (5–20 mmol/L) of NAC-induced G1 arrest in fibroblasts. NAC-induced G1 arrest is preceded by an increase in the steady-state levels of superoxide radical anion and a decrease in cyclin D1 protein levels [7]. In this study, we investigated the use of the topical NAC in prevention of postoperative pericardial adhesion in a rabbit model.
N. Colak et al. / Cardiovascular Pathology 22 (2013) 368–372
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Fig. 1. (A) Two weeks after the initial operation, moderate-to-severe pericardial adhesions, which were difficult to dissect out, were observed between the epicardium and the mediastinal tissues in group 2, (B) whereas the adhesion formation in Group 1 was significantly low between the epicardial and pericardial surfaces.
2. Material and methods The study protocol was approved by Fatih University of Medical Sciences, Ethics Committee for Animal Researches, Ankara, Turkey. Sixteen New Zealand white rabbits weighing 2.5 to 3.0 kg were anesthetized with 35-mg/kg ketamine hydrochloride and 5-mg/kg xylazine administered intramuscularly [8,9]. After disappearance of the pedal reflex in the hind limbs [10], the rabbits were placed in the supine position on a heated operating table and maintained at a temperature of 39°C by monitoring rectal temperature [11]. The pedal reflex was checked every 5 min throughout the surgical procedure. A venous line was established in the ear, and a saline solution was infused at a rate of 3 mg/kg/h. Prophylactic antibiotics (cefazolin sodium, 40 mg/kg) was given intravenously just before the operation. All had continuos two-lead electrocardiograph monitoring during the operative procedure. A pediatric facial mask in which oxygen gas flowed at a rate of 200 ml/min was placed on each rabbit [12]. The surgical procedure was performed in a sterilized fashion. After a midline muscle and skin incision was made over the sternum, the xiphoid process was carefully detached from the sternal part of the diaphragm. A median sternotomy was then performed; the median incision went straight down the xiphoid process toward the jugular notch of the sternum exactly along the midline of the sternum so that injury to the parietal pleura was avoided. Sternal retractors were then used to spread the sternal edges and maintain surgical exposure. The epicardium and parietal pericardium related to right ventricle atrium and right and left ventricle were abraded with 10 vertically reciprocal movement of a dry gauze to create local inflammation. The rabbits were divided into two groups: Group 1 was treated with the sponge, which was impregnated with NAC solution (ACT Ampul,10%, 300 mg/3 ml, Adeka, Turkey) and applied over the abraded epicardium for 5 min (n=8). Subsequently, this sponge was removed over the epicardium. Group 2 was the control, and the sponge, which was impregnated with 0.9% isotonic NaCl solution (serum fizyolojik 0.9% NaCl, 5 ml/ampul, Adeka, Turkey) was applied onto the surface of the abraded epicardium for 5 min (n=8). After 5 min, sponge was removed over the epicardium. Sternum was closed with three interrupted sutures using 3– 0 nylon with a taper point needle. Muscle layers and skin were then closed with continuous suture using 4–0 nylon with a cutting needle. The rabbits were allowed to recover. During the surgical procedure, all rabbits exhibited spontaneous respiration and loss of the pedal reflex. The rabbits were sacrificed 2 weeks after surgery with a lethal dose of pentobarbital (150 mg/kg) (Nembutol, İE Ulagay, Istanbul, Turkey). The heart and pericardium were removed en bloc. Specimens were fixed in 10% formaldehyde, embedded in paraffin and sectioned into
5-μm slices, which were stained with hematoxylin–eosin (H&E) to assess the inflammatory reaction, fibrosis degree, and check for remnants of the pericardial substitute in the two groups. 2.1. Macroscopic examination The surgeon, blinded to the treatment group, graded adhesion formation according to the score defined by Heydorn et al. [13]: Grade 0: no adhesions; Grade 1: light adhesions easily lysed by digital dissection; Grade 2: stronger adhesions; and Grade 3: dense adhesions requiring sharp dissection. One score was established for each animal. 2.2. Light microscopic examination Sections were examined using a 5× magnification objective lens by a pathologist blinded to the treatment group. The severity of the inflammatory reaction was based on the quantification of inflammatory cells (i.e., neutrophils, plasma cells, lymphocytes) and inflammatory foci and classified from 0 to 3, using an inflammatory reaction score derived from the one described by Lu et al. [14]: Grade 0: no cell infiltration; 1: sparse inflammatory cell infiltration; 2: focal inflammatory cell infiltration; and 3: diffuse inflammatory cell infiltration. Fibrosis was assessed with regard to its extent (0: limited; 1: great), density (0: loose; 1: dense), and thickness (0: thin; 1: thick). A composite end point was then created for each heart by summing the gross adhesion score (from 0 to 3) and the histological indices (inflammatory reaction score: from 0 to 3 and fibrosis score: from 0 to 3). 2.3. Power and sample size The sample size of the study was calculated with G*Power (G*Power Ver. 3.00.10, Franz Faul, Üniversität Kiel, Germany; www. psycho.uniduesseldorf.de/aap/projects/gpower/) statistical packages. The required sample size for 80% power, α=0.05 Type I error, β=0.20 Type II error, and f=0.70 effect size was calculated as 16 including eight New Zealand white rabbits in each group. To protect the study Table 1 Results of the adhesion, inflammation, and fibrosis scale
Adhesion Inflammation Fibrosis
Group 1 Median (range)
Group 2 Median (range)
P value
1 (1–2) 1.5 (1–3) 2 (1–2)
3 (2–3) 2.5 (1–3) 3 (2–3)
b0.001 0.083 0.007
Statistically significant results indicated in bold type.
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Table 2 Frequency of adhesion, fibrosis, and inflammation scores in the groups Group Adhesion
1
2
Fibrosis
1
2
Inflammation
1
2
n % % n % % n % % n % % n % % n % %
group adhesion group adhesion group fibrosis group fibrosis group inflammation group inflammation
None–Mild
Moderate–Severe
7 87.5 87.5 1 12.5 12.5 3 37.5 100.0 0 0.0 0.0 1 12.5 20.0 4 50.0 80.0
1 12.5 12.5 7 87.5 87.5 5 62.5 38.5 8 100.0 61.5 7 87.5 63.6 4 50.0 36.4
from potential lost to follow-ups, one more rabbit was included in each group, and the study was completed with a sample size of 18. 2.4. Statistical analysis Data coding and statistical analyses were conducted using Statistical Package for the Social Sciences (version 15; SPSS, Chicago, IL, USA). Following the entering of patient data into the computer, all the necessary diagnostic checks and corrections were performed. Conformity of the measured values to normal distribution was examined graphically and using a Shapiro–Wilks test. In presenting descriptive statistics, numbers and percentages were used for categorical variables and medians and ranges were used for nonnormally distributed variables. The Mann–Whitney U test was used for comparison of the median value in the groups. Chi-square was performed to assess the difference in the proportion of rabbits in the groups. Groups that are close to each other were combined and the 2×2 table chi-square test was created. Fisher's Exact Test was used for the comparison of these groups. Two-tailed Pb.05 was considered statistically significant. 3. Results All animals tolerated the procedure with no apparent postoperative complications. Results were analyzed in terms of macroscopic and microscopic findings. 3.1. Macroscopic findings A total of 16 rabbits were evaluated for grading of adhesions by macroscopic finding. In seven cases of NAC group, adhesions were
split by blunt dissection (Fig. 1A). At the opposite, the seven of the control group (epicardial abrasion and 0.9% NaCl solution) was associated with tight adherences to the sternum and the rest of the pericardium requiring sharp dissection (Fig. 1B). There was statistically significant difference between the NAC group and control group in terms of the adhesion score [Group 1 vs. 2; 0.5 (0–2) vs. 2 (1–3), P= .003] (Table 1). When the prevalence of adhesion was compared, there was a positive trend in the odds ratio for moderate-to-severe adhesion in the Group 2 [Group 1 vs. 2; 12.5% vs. 87.5%, respectively] (Table 2). When the prevalence of adhesion was compared, there was a positive trend in the odds ratio for moderate-to-severe adhesion in the Group 2 (P=.013) (Table 2). Results of the adhesion, inflammation, and fibrosis scale are shown in Fig. 2. 3.2. Microscopic findings There was no difference between groups in terms of the degree of inflammation (P=.083) (Table 1). When they compare for the prevalence of inflammation, although moderate and severe inflammation was less observed in the Group 1, there was no statistically significant difference between the groups (P=.141) (Table 2). A statistically significant difference between the NAC group and control group in terms of the severity of fibrosis was observed (P= .007) (Table 1). However, despite the presence of 37.5% of none-tominimal fibrosis in the Group 1, no significant difference between groups in terms of the prevalence of fibrosis was detected (P=.100) (Fig. 3A, B). 4. Discussion This study showed application in the pericardial cavity with these NAC (a sulfidryl containing thiol antioxidant) after abrasive injury resulted in significant reduction in mean adhesion scores compared to irrigation of the pericardium with only 0.9% isotonic NaCl solution. In addition and of potentially even greater histopathological importance, fibrosis and inflammation scores were essentially reduced when this NAC solution was used whereas both of these scores (including inflammation and fibrosis) were higher in animals treated with 0.9% isotonic NaCl solution. The formation of postoperative adhesion is an inevitable sequel to surgical intervention and as part of the healing process. Histologic examination of pericardial tissue from animals undergoing cardiac surgical procedures indicates that damage to the mesothelium (especially by abrasion) is adhesiogenic [15,16]. Inflammatory responses are important in the pathogenesis of adhesion formation. Reduction in tissue oxygenation due to ischemia is known to be involved in adhesion formation. Free radicals, significantly produced after endothelial tissue damage, are able to react rapidly with oxygen and so exacerbate the
Fig. 2. The distribution between groups according to adhesion, inflammation and fibrosis scores.
N. Colak et al. / Cardiovascular Pathology 22 (2013) 368–372
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Fig. 3. (A) Histological section of the control group (Group 2); dense deposits of fibrin in between infiltration of the inflammatory cells which including the intensive lymphoplasmacytoid cells and the polymorphonuclear leukocytes histiocytes, proliferation of capillary vessels, fibroblastic proliferation, and fibrosis were observed in the pericardial tissues (H&E stain × 100); (B) Histological section of the study group (Group 1); infiltration of the focal inflammatory cells, a slight proliferation of capillary vessels, and focal fibroblastic proliferation were showed in the pericardial tissues (H&E stain × 100) (black arrow indicates the inflammatory cell; white arrow indicates the fibrin deposits).
oxygen deficit [17]. Moreover, free radicals such as superoxides, peroxides, and hydroxyl radicals are potential oxidizers of polyunsaturated fatty acids resulting in peroxidation of cellular membrane lipids and increased vascular permeability [18]. This leads to the formation of serosanguineous exuda, which in turn initiates adhesion formation. Subsequently, if these initial adhesions are not lysed, they are organized into fibrous adhesions by activated fibroblasts. However, in a state of an imbalance between fibrin deposition and dissolution, deposited fibrin may persist, and fibrinous adhesions may develop [17]. It is an appreciation of this pathophysiology and an incorporation of its principles into methods used to overcome problems of adhesions that may enable an appropriate manipulation of adhesion formation. Unfortunately, despite continuous advances and research, an ideal method to prevent postoperative pericardial adhesion formation has not been confirmed. In the present study, NAC was used at reduced pericardial injury due to abrasion, which would initiate adhesion formation. NAC, the acetylated variant of the amino acid L-cysteine, is an excellent source of SH groups and is converted in the body into metabolites capable of stimulating GSH synthesis, promoting detoxification, and acting directly as free radical scavengers [6]. The antioxidant effects of NAC were examined using different organ ischemia–reperfusion models [19,20]. A recent study also showed varying doses of NAC-induced G1 arrest in fibroblasts. NAC-induced G1 arrest is preceded by an increase in the steady-state levels of superoxide radical anion and a decrease in cyclin D1 protein levels. Furthermore, their results were showed that NAC's antioxidant property could be, in part, due to the increased superoxide dismutase activity [7]. Also observed in our study, reduction of adhesion and fibrosis may be due to that activity of NAC. In addition, use of the topical NAC solution, which is a sulfidryl-containing thiol, may create a physical barrier to maintain separation of injured tissue during the healing process such as other thiol-containing molecules (e.g., Carbylan-S). A number of antiadhesive interventions have been developed, and many have been tested clinically and experimentally in cardiac applications. Challenges of current materials and agents include technical difficulties with surgical application, such as the need for complete hemostasis and the requirement to secure some barriers with sutures that may actually promote fibrosis. Xenogeneic and synthetic pericardial substitutes also have been shown to be associated with inflammation, epicardial reaction, fibrous encapsulation, and calcification and have not demonstrated consistent antiadhesive effectiveness [21,22]. While systemic antiinflammatory drugs and topical application of fibrinolytic agents have been shown to reduce adhesion formation, both modalities have the untoward side effects of bruising, bleeding, and impairment of wound healing [23]. Application of NAC in the pericardial cavity has not seen the
untoward side effect in this study. In addition, this agent is nontoxic, does not exhibit an inflammatory response, and does not interfere with normal wound healing [19,24]. In conclusion, NAC solution has been demonstrated to be effective in reducing the formation of postoperative adhesions in an experimentally relevant rabbit adhesion model. From this study, the mechanism by which NAC solution reduces pericardial adhesion formation may be related to its antifibrotic activity and protection of fragile serosal surfaces from abrasive trauma during surgery (physical barrier potential). The most appropriate dose or delivery method for clinical application needs to be determined.
References [1] Loop FD, Cosgrove DM, Kramer JR. Late clinical and arteriographic results in 500 coronary artery reoperations. J Thorac Cardiovasc Surg 1981;81:675–84. [2] Diamond P, DeCherney AH. Pathogenesis of adhesion formation/reformation: application to reproductive pelvic surgery. Microsurgery 1987;8:103. [3] Edwards GA, Glattauer V, Nash TJ, et al. In vivo evaluation of a collagenous membrane as an absorbable adhesion barrier. J Biomed Mater Res 1997;34:291. [4] Matsuda S, Se N, Iwata H, Ikada Y. Evaluation of the antiadhesion potential of UV cross-linked gelatin films in a rat abdominal model. Biomaterials 2002;23:2901. [5] Kelly GS. Clinical applications of N-acetylcysteine. Altern Med Rev 1998;3:114–27. [6] Aruoma OI, Halliwell B, Hoey BM, Butler J. The antioxidant action of Nacetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 1989;6(6):593–7. [7] Menon SG, Sarsour EH, Kalen AL, Venkataraman S, Hitchler MJ, Domann FE, et al. Superoxide signaling mediates N-acetyl-L-cysteine-induced G1 arrest: regulatory role of cyclin D1 and manganese superoxide dismutase. Cancer Res 2007;67(13): 6392–9. [8] White GL, Holmes DD. A comparison of ketamine and the combination ketaminexylazine for effective surgical anesthesia in the rabbit. Lab Anim Sci 1976;26:804. [9] Green CJ, Knight J, Precious S, Simpkin S. Ketamine alone and combined with diazepam or xylazine in laboratory animals: a 10 year experience. Lab Anim 1981;15:163. [10] Flecknell PA, John M, Mitchell M, Shurey C. Injectable anaesthetic techniques in 2 species of gerbil (Meriones libycus and Meriones unguiculatus). Lab Anim 1983;17:118. [11] Peeters ME, Gil D, Teske E, Eyzenbach V, vd Brom WE, Lumeij JT, et al. Four methods for general anaesthesia in the rabbit: a comparative study. Lab Anim 1988;22:355. [12] Fujita M, Morimoto Y, Ishihara M, Shimizu M, Takase B, Maehara T, et al. A new rabbit model of myocardial infarction without endotracheal intubation. J Surg Res 2004;116(1):124–8. [13] Heydorn WH, Daniel JS, Wade CE. A new look at pericardial substitutes. J Thorac Cardiovasc Surg 1987;94:291–6. [14] Lu JH, Chang Y, Sung HW, Chiu YT, Yang PC, Hwang B. Heparinization on pericardial substitutes can reduce adhesion and epicardial inflammation in the dog. J Thorac Cardiovasc Surg 1998;115:1111–20. [15] Burns JW, Skinner K, Colt J, Sheidlin A, Bronson R, Yaacobi Y, et al. Prevention of tissue injury and postsurgical adhesions by precoating tissues with hyaluronic acid solutions. J Surg Res 1995;59:644. [16] Cliff WJ, Grobety J, Ryan GB. Postoperative pericar-dial adhesions: the role of mild serosal injury and spilled blood. J Thoracic Cardiovasc Surg 1973;65:744. [17] Binda MM, Molinas CR, Koninckx PR. Reactive oxygen species and adhesion formation: clinical implications in adhesion prevention. Hum Reprod 2003;18:2503–7. [18] Kagoma P, Burger SN, Seifter E, Levenson SM, Demetriou AA. The effect of vitamin E on experimentally induced peritoneal adhesions in mice. Arch Surg 1985;120: 949–51.
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[19] Cakir O, Oruc A, Kaya S, Eren N, Yildiz F, Erdinc L. N-acetylcysteine reduces lung reperfusion injury after deep hypothermia and total circulatory arrest. J Card Surg 2004;19(3):221–5. [20] Inci I, Erne B, Arni S, Jungraithmayr W, Inci D, Hillinger S, et al. Prevention of primary graft dysfunction in lung transplantation by N-acetylcysteine after prolonged cold ischemia. J Heart Lung Transplant 2010;29:1293–301. [21] Shapira N, Gordon CI, Lemole GM. Occlusion of aortocoronary vein grafts in association with bovine pericardium. Am J Cardiovasc Pathol 1990;3: 87–90.
[22] Urschel HC, Razzuk MA, Gardner M. Coronary artery bypass occlusion second to postcardiotomy syndrome. Ann Thorac Surg 1976;22:528–31. [23] Konertz WF, Kostelka M, Mohr FW, Hetzer R, Hübler M, Ritter J, et al. Reducing the incidence and severity of pericardial adhesions with a sprayable polymeric matrix. Ann Thorac Surg 2003;76:1270–4. [24] Aktunc E, Ozacmak VH, Ozacmak HS, Barut F, Buyukates M, Kandemir O, et al. N-acetyl cysteine promotes angiogenesis and clearance of free oxygen radicals, thus improving wound healing in an alloxan-induced diabetic mouse model of incisional wound. Clin Exp Dermatol 2010;35(8):902–9.
Contents lists available at ScienceDirect
Cardiovascular Pathology
Orginal Article
Effect of topical N-acetylcysteine in the prevention of postoperative pericardial adhesion formation in a rabbit model Necmettin Colak a,⁎, Yunus Nazli a, Mehmet Fatih Alpay a, Omer Nuri Aksoy a, Ismail Olgun Akkaya a, Reyhan Bayrak b, Omer Cakir a a b
Department of Cardiovascular Surgery, University of Fatih, Faculty of Medicine, Ankara, Turkey Department of Pathology, University of Fatih, Faculty of Medicine, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 3 July 2012 Received in revised form 21 January 2013 Accepted 6 February 2013 Keywords: Cardiac operation N-acetylcysteine Pericardial adhesion
a b s t r a c t Background: N-acetylcysteine (NAC), a precursor of reduced glutathione, has been in clinical use primarily as a mucolytic. In addition, NAC is well known for their free radical scavenging and antioxidant properties. Increasing of reactive oxygen products occurring during cardiac surgery can play an important role in postoperative adhesion formation. We investigated to the efficacy of the NAC for postoperative pericardial adhesions. Methods: Sixteen New Zealand white rabbits (2.5–3 kg) were used and categorized into two groups including study (use of NAC) and control groups. In both groups, the pericardium was opened longitudinally, and the exposed epicardial surfaces were abraded with dry gauze. The rabbits were divided into two groups: Group 1 was treated with the sponge, which impregnated with NAC solution, (10%, 300 mg/3 ml) and applied over the abraded epicardium for 5 min (n=8). Group 2 was the control, and the sponge, which was impregnated with 3-ml isotonic NaCl solution (0.9%), was applied onto the surface of the abraded epicardium for 5 min (n=8). After a period of 2 weeks, the animals were sacrificed. The scores of adhesion were graded by macroscopic examination, and the pericardial tissues were analyzed microscopically in point of inflammation and fibrosis. Results: In Group 1, the adhesion scores were significantly lower compared with the control group [Group 1 vs. 2; 1 (1–2) vs. 3 (2–3), Pb.001]. No significant difference was found between the groups in terms of the severity of inflammation [Group 1 vs. 2; 1.5 (1–3) vs. 2.5 (1–3), P=.083]. There was a difference between groups in terms of the degree of fibrosis [Group 1 vs. 2; 2 (1–2) vs. 3 (2–3), P=.007]. Conclusions: The use of NAC for preventing postoperative pericardial adhesions was reduced to adhesion and fibrosis scores in an experimental rabbit model. There was no statistically significant difference between groups in terms of inflammatory scores. The NAC effectively prevented the formation of pericardial adhesion. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Cardiac surgical procedures routinely results in the development of extensive fibrous adhesions. The condition may be general or localized and may involve adhesion between the epicardium and the pericardium, sternum, pleura, or surrounding tissues such as the diaphragm or other mediastinal structures, as a result of an inflammatory process. The presence of these adhesions not only adds to the operating time but also increases the risk of damage to the heart, great vessels, and previously placed grafts [1]. Especially, retrosternal adhesions resulting from previous surgery continue to be a challenge during resternotomy. Different techniques were applied to reduce or prevent development of postoperative adhesions in the The financial support from an institution was not taken in this study. ⁎ Corresponding author. Alparslan Turkes caddesi No:57, 06510, Emek/Ankara, Turkey. Tel.: +90 3122035187; fax: +90 3122213670. E-mail address: [email protected] (N. Colak). 1054-8807/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.carpath.2013.02.001
retrosternal spaces and mediastinal structures. Fibrinolytic agents, anticoagulants, antiinflammatory agents, several natural and synthetic physical barriers, and antibiotics have been tried with variable success [2–4]. Despite continuous advances and research, an ideal method to prevent postoperative pericardial adhesion formation has not been confirmed. N-acetylcysteine (NAC), the acetylated variant of the amino acid L-cysteine, is an excellent source of sulfhydryl (SH) groups and is converted in the body into metabolites capable of stimulating glutathione (GSH) synthesis, promoting detoxification, and acting directly as free radical scavengers [5]. The antioxidant activities of NAC have been reported in previous studies [6]. In addition, another study was reported varying doses (5–20 mmol/L) of NAC-induced G1 arrest in fibroblasts. NAC-induced G1 arrest is preceded by an increase in the steady-state levels of superoxide radical anion and a decrease in cyclin D1 protein levels [7]. In this study, we investigated the use of the topical NAC in prevention of postoperative pericardial adhesion in a rabbit model.
N. Colak et al. / Cardiovascular Pathology 22 (2013) 368–372
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Fig. 1. (A) Two weeks after the initial operation, moderate-to-severe pericardial adhesions, which were difficult to dissect out, were observed between the epicardium and the mediastinal tissues in group 2, (B) whereas the adhesion formation in Group 1 was significantly low between the epicardial and pericardial surfaces.
2. Material and methods The study protocol was approved by Fatih University of Medical Sciences, Ethics Committee for Animal Researches, Ankara, Turkey. Sixteen New Zealand white rabbits weighing 2.5 to 3.0 kg were anesthetized with 35-mg/kg ketamine hydrochloride and 5-mg/kg xylazine administered intramuscularly [8,9]. After disappearance of the pedal reflex in the hind limbs [10], the rabbits were placed in the supine position on a heated operating table and maintained at a temperature of 39°C by monitoring rectal temperature [11]. The pedal reflex was checked every 5 min throughout the surgical procedure. A venous line was established in the ear, and a saline solution was infused at a rate of 3 mg/kg/h. Prophylactic antibiotics (cefazolin sodium, 40 mg/kg) was given intravenously just before the operation. All had continuos two-lead electrocardiograph monitoring during the operative procedure. A pediatric facial mask in which oxygen gas flowed at a rate of 200 ml/min was placed on each rabbit [12]. The surgical procedure was performed in a sterilized fashion. After a midline muscle and skin incision was made over the sternum, the xiphoid process was carefully detached from the sternal part of the diaphragm. A median sternotomy was then performed; the median incision went straight down the xiphoid process toward the jugular notch of the sternum exactly along the midline of the sternum so that injury to the parietal pleura was avoided. Sternal retractors were then used to spread the sternal edges and maintain surgical exposure. The epicardium and parietal pericardium related to right ventricle atrium and right and left ventricle were abraded with 10 vertically reciprocal movement of a dry gauze to create local inflammation. The rabbits were divided into two groups: Group 1 was treated with the sponge, which was impregnated with NAC solution (ACT Ampul,10%, 300 mg/3 ml, Adeka, Turkey) and applied over the abraded epicardium for 5 min (n=8). Subsequently, this sponge was removed over the epicardium. Group 2 was the control, and the sponge, which was impregnated with 0.9% isotonic NaCl solution (serum fizyolojik 0.9% NaCl, 5 ml/ampul, Adeka, Turkey) was applied onto the surface of the abraded epicardium for 5 min (n=8). After 5 min, sponge was removed over the epicardium. Sternum was closed with three interrupted sutures using 3– 0 nylon with a taper point needle. Muscle layers and skin were then closed with continuous suture using 4–0 nylon with a cutting needle. The rabbits were allowed to recover. During the surgical procedure, all rabbits exhibited spontaneous respiration and loss of the pedal reflex. The rabbits were sacrificed 2 weeks after surgery with a lethal dose of pentobarbital (150 mg/kg) (Nembutol, İE Ulagay, Istanbul, Turkey). The heart and pericardium were removed en bloc. Specimens were fixed in 10% formaldehyde, embedded in paraffin and sectioned into
5-μm slices, which were stained with hematoxylin–eosin (H&E) to assess the inflammatory reaction, fibrosis degree, and check for remnants of the pericardial substitute in the two groups. 2.1. Macroscopic examination The surgeon, blinded to the treatment group, graded adhesion formation according to the score defined by Heydorn et al. [13]: Grade 0: no adhesions; Grade 1: light adhesions easily lysed by digital dissection; Grade 2: stronger adhesions; and Grade 3: dense adhesions requiring sharp dissection. One score was established for each animal. 2.2. Light microscopic examination Sections were examined using a 5× magnification objective lens by a pathologist blinded to the treatment group. The severity of the inflammatory reaction was based on the quantification of inflammatory cells (i.e., neutrophils, plasma cells, lymphocytes) and inflammatory foci and classified from 0 to 3, using an inflammatory reaction score derived from the one described by Lu et al. [14]: Grade 0: no cell infiltration; 1: sparse inflammatory cell infiltration; 2: focal inflammatory cell infiltration; and 3: diffuse inflammatory cell infiltration. Fibrosis was assessed with regard to its extent (0: limited; 1: great), density (0: loose; 1: dense), and thickness (0: thin; 1: thick). A composite end point was then created for each heart by summing the gross adhesion score (from 0 to 3) and the histological indices (inflammatory reaction score: from 0 to 3 and fibrosis score: from 0 to 3). 2.3. Power and sample size The sample size of the study was calculated with G*Power (G*Power Ver. 3.00.10, Franz Faul, Üniversität Kiel, Germany; www. psycho.uniduesseldorf.de/aap/projects/gpower/) statistical packages. The required sample size for 80% power, α=0.05 Type I error, β=0.20 Type II error, and f=0.70 effect size was calculated as 16 including eight New Zealand white rabbits in each group. To protect the study Table 1 Results of the adhesion, inflammation, and fibrosis scale
Adhesion Inflammation Fibrosis
Group 1 Median (range)
Group 2 Median (range)
P value
1 (1–2) 1.5 (1–3) 2 (1–2)
3 (2–3) 2.5 (1–3) 3 (2–3)
b0.001 0.083 0.007
Statistically significant results indicated in bold type.
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Table 2 Frequency of adhesion, fibrosis, and inflammation scores in the groups Group Adhesion
1
2
Fibrosis
1
2
Inflammation
1
2
n % % n % % n % % n % % n % % n % %
group adhesion group adhesion group fibrosis group fibrosis group inflammation group inflammation
None–Mild
Moderate–Severe
7 87.5 87.5 1 12.5 12.5 3 37.5 100.0 0 0.0 0.0 1 12.5 20.0 4 50.0 80.0
1 12.5 12.5 7 87.5 87.5 5 62.5 38.5 8 100.0 61.5 7 87.5 63.6 4 50.0 36.4
from potential lost to follow-ups, one more rabbit was included in each group, and the study was completed with a sample size of 18. 2.4. Statistical analysis Data coding and statistical analyses were conducted using Statistical Package for the Social Sciences (version 15; SPSS, Chicago, IL, USA). Following the entering of patient data into the computer, all the necessary diagnostic checks and corrections were performed. Conformity of the measured values to normal distribution was examined graphically and using a Shapiro–Wilks test. In presenting descriptive statistics, numbers and percentages were used for categorical variables and medians and ranges were used for nonnormally distributed variables. The Mann–Whitney U test was used for comparison of the median value in the groups. Chi-square was performed to assess the difference in the proportion of rabbits in the groups. Groups that are close to each other were combined and the 2×2 table chi-square test was created. Fisher's Exact Test was used for the comparison of these groups. Two-tailed Pb.05 was considered statistically significant. 3. Results All animals tolerated the procedure with no apparent postoperative complications. Results were analyzed in terms of macroscopic and microscopic findings. 3.1. Macroscopic findings A total of 16 rabbits were evaluated for grading of adhesions by macroscopic finding. In seven cases of NAC group, adhesions were
split by blunt dissection (Fig. 1A). At the opposite, the seven of the control group (epicardial abrasion and 0.9% NaCl solution) was associated with tight adherences to the sternum and the rest of the pericardium requiring sharp dissection (Fig. 1B). There was statistically significant difference between the NAC group and control group in terms of the adhesion score [Group 1 vs. 2; 0.5 (0–2) vs. 2 (1–3), P= .003] (Table 1). When the prevalence of adhesion was compared, there was a positive trend in the odds ratio for moderate-to-severe adhesion in the Group 2 [Group 1 vs. 2; 12.5% vs. 87.5%, respectively] (Table 2). When the prevalence of adhesion was compared, there was a positive trend in the odds ratio for moderate-to-severe adhesion in the Group 2 (P=.013) (Table 2). Results of the adhesion, inflammation, and fibrosis scale are shown in Fig. 2. 3.2. Microscopic findings There was no difference between groups in terms of the degree of inflammation (P=.083) (Table 1). When they compare for the prevalence of inflammation, although moderate and severe inflammation was less observed in the Group 1, there was no statistically significant difference between the groups (P=.141) (Table 2). A statistically significant difference between the NAC group and control group in terms of the severity of fibrosis was observed (P= .007) (Table 1). However, despite the presence of 37.5% of none-tominimal fibrosis in the Group 1, no significant difference between groups in terms of the prevalence of fibrosis was detected (P=.100) (Fig. 3A, B). 4. Discussion This study showed application in the pericardial cavity with these NAC (a sulfidryl containing thiol antioxidant) after abrasive injury resulted in significant reduction in mean adhesion scores compared to irrigation of the pericardium with only 0.9% isotonic NaCl solution. In addition and of potentially even greater histopathological importance, fibrosis and inflammation scores were essentially reduced when this NAC solution was used whereas both of these scores (including inflammation and fibrosis) were higher in animals treated with 0.9% isotonic NaCl solution. The formation of postoperative adhesion is an inevitable sequel to surgical intervention and as part of the healing process. Histologic examination of pericardial tissue from animals undergoing cardiac surgical procedures indicates that damage to the mesothelium (especially by abrasion) is adhesiogenic [15,16]. Inflammatory responses are important in the pathogenesis of adhesion formation. Reduction in tissue oxygenation due to ischemia is known to be involved in adhesion formation. Free radicals, significantly produced after endothelial tissue damage, are able to react rapidly with oxygen and so exacerbate the
Fig. 2. The distribution between groups according to adhesion, inflammation and fibrosis scores.
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Fig. 3. (A) Histological section of the control group (Group 2); dense deposits of fibrin in between infiltration of the inflammatory cells which including the intensive lymphoplasmacytoid cells and the polymorphonuclear leukocytes histiocytes, proliferation of capillary vessels, fibroblastic proliferation, and fibrosis were observed in the pericardial tissues (H&E stain × 100); (B) Histological section of the study group (Group 1); infiltration of the focal inflammatory cells, a slight proliferation of capillary vessels, and focal fibroblastic proliferation were showed in the pericardial tissues (H&E stain × 100) (black arrow indicates the inflammatory cell; white arrow indicates the fibrin deposits).
oxygen deficit [17]. Moreover, free radicals such as superoxides, peroxides, and hydroxyl radicals are potential oxidizers of polyunsaturated fatty acids resulting in peroxidation of cellular membrane lipids and increased vascular permeability [18]. This leads to the formation of serosanguineous exuda, which in turn initiates adhesion formation. Subsequently, if these initial adhesions are not lysed, they are organized into fibrous adhesions by activated fibroblasts. However, in a state of an imbalance between fibrin deposition and dissolution, deposited fibrin may persist, and fibrinous adhesions may develop [17]. It is an appreciation of this pathophysiology and an incorporation of its principles into methods used to overcome problems of adhesions that may enable an appropriate manipulation of adhesion formation. Unfortunately, despite continuous advances and research, an ideal method to prevent postoperative pericardial adhesion formation has not been confirmed. In the present study, NAC was used at reduced pericardial injury due to abrasion, which would initiate adhesion formation. NAC, the acetylated variant of the amino acid L-cysteine, is an excellent source of SH groups and is converted in the body into metabolites capable of stimulating GSH synthesis, promoting detoxification, and acting directly as free radical scavengers [6]. The antioxidant effects of NAC were examined using different organ ischemia–reperfusion models [19,20]. A recent study also showed varying doses of NAC-induced G1 arrest in fibroblasts. NAC-induced G1 arrest is preceded by an increase in the steady-state levels of superoxide radical anion and a decrease in cyclin D1 protein levels. Furthermore, their results were showed that NAC's antioxidant property could be, in part, due to the increased superoxide dismutase activity [7]. Also observed in our study, reduction of adhesion and fibrosis may be due to that activity of NAC. In addition, use of the topical NAC solution, which is a sulfidryl-containing thiol, may create a physical barrier to maintain separation of injured tissue during the healing process such as other thiol-containing molecules (e.g., Carbylan-S). A number of antiadhesive interventions have been developed, and many have been tested clinically and experimentally in cardiac applications. Challenges of current materials and agents include technical difficulties with surgical application, such as the need for complete hemostasis and the requirement to secure some barriers with sutures that may actually promote fibrosis. Xenogeneic and synthetic pericardial substitutes also have been shown to be associated with inflammation, epicardial reaction, fibrous encapsulation, and calcification and have not demonstrated consistent antiadhesive effectiveness [21,22]. While systemic antiinflammatory drugs and topical application of fibrinolytic agents have been shown to reduce adhesion formation, both modalities have the untoward side effects of bruising, bleeding, and impairment of wound healing [23]. Application of NAC in the pericardial cavity has not seen the
untoward side effect in this study. In addition, this agent is nontoxic, does not exhibit an inflammatory response, and does not interfere with normal wound healing [19,24]. In conclusion, NAC solution has been demonstrated to be effective in reducing the formation of postoperative adhesions in an experimentally relevant rabbit adhesion model. From this study, the mechanism by which NAC solution reduces pericardial adhesion formation may be related to its antifibrotic activity and protection of fragile serosal surfaces from abrasive trauma during surgery (physical barrier potential). The most appropriate dose or delivery method for clinical application needs to be determined.
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