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A randomized controlled study of the efficacy of misoprostol and hyaluronic acid in preventing adhesion formation after gynecological surgery: a rat uterine horn model
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A randomized controlled study of the efficacy of misoprostol and hyaluronic acid in preventing adhesion formation after gynecological surgery: a rat uterine horn model Cihan Kaya a,*, Nurten Sever b, Hu¨seyin Cengiz c, S¸u¨kru¨ Yıldız c, Murat Ekin c, Levent Yas¸ar c Van O¨zalp State Hospital, Clinic of Obstetrics and Gynecology, Turkey Bakirkoy Dr Sadi Konuk Training and Research Hospital, Department of Pathology, Turkey c Bakirkoy Dr Sadi Konuk Training and Research Hospital, Department of Obstetrics and Gynecology, Turkey a
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 December 2013 Received in revised form 3 February 2014 Accepted 14 February 2014
Objective: To investigate the effect of misoprostol in the reduction of adhesion formation after gynecological surgery. Study design: A double blind, randomized controlled experimental study was designed. Twenty-one female Wistar Hannover rats were divided into three groups as control, misoprostol and Hyalobarrier1 groups. A uterine horn adhesion model was created. After anesthesia induction, 1.5–2 cm injuries were made to the each uterine horn by cautery. The control group received no special medications except for the standard surgical procedure. The misoprostol group received 10 mcg/kg misoprostol in addition to the standard surgical procedure, and the Hyalobarrier1 group received 1 cm3 ready-for-use Hyalobarrier1 gel intraperitoneally in addition to the standard surgical procedure. After 14 days from the first surgical procedure, adhesion scores were evaluated. Results: The extent (p < 0.001), severity (p < 0.001), degree (p < 0.001) and total adhesion score (p < 0.001) values of the control group were statistically higher than the values of misoprostol and Hyalobarrier1 groups. The inflammation score value of misoprostol group was statistically lower than control and Hyalobarrier1 groups (p < 0.001). Conclusion: In this study, we have found a new therapeutic potential of misoprostol that may be useful in preventing pelvic adhesion and reducing inflammation scores. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Surgical adhesions Hyaluronic acid Misoprostol PGE1 TGF b1
1. Introduction Postoperative intra-abdominal adhesions occur in 50–95% of women who undergo gynecological surgery [1]. The development of peritoneal adhesions following abdominal and pelvic surgery leads to clinical problems, including intestinal obstruction, infertility, and chronic pelvic pain [2]. Adhesions remain a potential problem for future surgical procedures, increasing health expenses and hospital readmission rates, and reducing the quality of life of the patient [3]. There are several described causes of adhesions, such as mechanical trauma, ischemia caused by sutures or electrocautery, presence of foreign bodies, tissue desiccation,
¨ zalp State Hospital, Clinic of Obstetrics and * Corresponding author at: Van O ¨ zalp, Van, Turkey. Gynecology, Cumhuriyet Mh., No. 3 Merkez O Tel.: +90 212 414 73 72. E-mail address: [email protected] (C. Kaya).
and infection [4]. Furthermore, during peritoneal healing following surgery, the increase in peritoneal fluid, cytokines, proteins and fibrin formation can promote adhesion formation [5,6]. Currently, there are various methods of preventing adhesion formation such as the use of barrier materials, non-steroid antiinflammatory drugs, calcium channel blockers, corticosteroids, vitamin E, antihistamines, metformin, melatonin, progesterone, estrogen, gonadotrophin-releasing hormone (GnRH)-agonists and antagonists, anticoagulants, fibrinolytic agents, and antibiotics [7– 10]. Hyalobarrier1 gel is a highly viscous gel derived from hyaluronan, obtained through an auto-crosslinking process, and used as an adhesion prevention agent in a variety of surgical procedures [11–13]. Hyalobarrier1 gel can reduce the incidence and severity of postoperative adhesions in cases of severe uterine damage caused by laparotomy or laparoscopic myomectomies [14,15]. The gel can also be easily applied during laparoscopic and hysteroscopic procedures.
http://dx.doi.org/10.1016/j.ejogrb.2014.02.033 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Kaya C, et al. A randomized controlled study of the efficacy of misoprostol and hyaluronic acid in preventing adhesion formation after gynecological surgery: a rat uterine horn model. Eur J Obstet Gynecol (2014), http://dx.doi.org/ 10.1016/j.ejogrb.2014.02.033
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Misoprostol, a synthetic analog of prostaglandin E1, is used as an anti-ulcer drug that increases gastric mucus secretion and reduces gastric acid secretion [16]. Misoprostol is also used offlabel by obstetricians for the induction of first trimester abortion [17]. Misoprostol has been shown to alter enzymatic processes that cause degradation and remodeling of collagen fibers [18]. Nevertheless, no in vivo studies have demonstrated that misoprostol can reduce adhesion formation. Misoprostol is commercially available as Cytotec1 (Ali Raif I˙lac¸, I˙stanbul, Tu¨rkiye) in a pharmaceutical complex with hydroxypropyl methylcellulose, microcrystalline cellulose, sodium starch glycolate, and hydrogenated castor oil [19]. In this study, we aimed to investigate the effect of the intraperitoneal use of misoprostol in the prevention of adhesions by reducing collagen synthesis, which plays a major role in wound healing and scar formation, compared with that of Hyalobarrier1 gel, which is known to prevent adhesions in clinical practice. 2. Materials and methods The study was approved by the local ethical review board of the Bagcilar Training and Research Hospital in Istanbul, Turkey, for the use of laboratory animals, and was performed at the experimental surgery training and education center of the same hospital (approval number: 2013–17). 2.1. Animal maintenance and treatment Rats weighing approximately 180–260 g were housed under the institutional review board’s guidelines for animal care using a day cycle of 14 h of light with free access to food and water. A power analysis was performed to calculate the minimum sample size required for the study (alpha error = 0.05 and 1-beta = 0.8) and at least 12 uterine horns were required for each study group. As 10% of the animals are lost during the procedures, we included in total 14 horns. The rats were randomly assigned to three study groups, each consisting of seven rats, using computer-based randomization. Twenty-one mature, nonpregnant, female Wistar Hannover rats (aged 8–10 weeks) were used as models for the experimental induction of postoperative uterine horn adhesion, as defined by Kelekci et al. [20]. Animals were anesthetized using 50 mg/kg of 10% ketamine hydrochloride (Ketasol; Richter Pharma AG, Wels, Austria) intramuscularly (IM) and 5 mg/kg of 2% xylazine (Rompun; Bayer Health Care LCC, Kansas, KS) IM. Prior to the operation, the abdominal skin was shaved and disinfected using 10% povidone-iodine solution (Batticon; Adeka Laboratories, Istanbul, Turkey). A 3-cm midline incision was made, and the uterine horns were exposed. Seven standard lesions were inflicted in the antimesenteric surfaces of each uterine horn using a 10 W bipolar cautery (Bovie1, FL, USA). An area of 1.5 cm 1.5 cm between the two main vascular branches of the lower side wall at the level of each uterine horn was cauterized to produce an opposing lesion. The first group of rats served as the control group and received no special medication. The second group (misoprostol group) received 10 mcg/kg misoprostol intraperitoneally (IP) using a diluted Cytotec1 200 mcg tablet (Ali Raif I˙lac¸, I˙stanbul, Tu¨rkiye). The third group (Hyalobarrier1 gel group) received 1 mL of readyto-use Hyalobarrier1 gel (Fidia Advanced Biopolymers S.r.I Abano Terme, Italy) IP. The abdominal incision was closed in two layers with a 4-0 polyglycolic acid suture (VicrylTM; Johnson and Johnson Medical Limited, Ethicon Limited, UK) for the peritoneum and 3-0 polyglactin suture for the skin. After the animals recovered from surgery, they were housed separately under controlled temperatures of 22 2 8C and a 14 h light cycle with food and water ad libitum. The surgery was limited to approximately 15 min for each rat to prevent tissue drying at room
temperature. All the surgical procedures were performed by the same researchers. 2.2. Tissue sample collection and histopathology Following the 14-day recovery period, the rats were sacrificed by cervical dislocation. The adhesion areas were evaluated and graded macroscopically by two observers who were blinded to the study groups. The extent and severity of the adhesions were evaluated using an established scoring system [21]. The extent of adhesion spread to the uterine horn defect was scored as follows: 0, no uterine adhesion; 1, 1–25% involvement, 2, 26–50% involvement, 3, 51–75% involvement, and 4, 76–100% involvement. Adhesions were further characterized on gross examination according to the severity of attachment between the uterine horns and other abdominal organs as follows: 0, no adhesion, 1, filmy avascular, 2, vascular or opaque, and 3, cohesive. The degree of adhesion formation was evaluated and scored using the following; 0, no adhesion; 1, the adhesion could be separated from the tissue with gentle traction, 2, the adhesion could be separated from the tissue with moderate traction, 3, the adhesion could be separated from the tissue by sharp dissection. The sum of the three parameters was used as the total score for each group. Tissue samples were obtained from all serosal surfaces where adhesions had developed. One histologist evaluated all tissues and was blinded to the origin of the samples. Tissues were graded for levels of inflammation and fibrosis using previously published grading scales. For histological analyses, the excised tissues were fixed in 10% buffered formalin solution for 24 h. After fixation, a routine tissue-processing procedure was performed, and sample tissues were embedded in paraffin. Paraffin wax blocks were cut into 4-mm thick sections. Prepared sections were then stained with hematoxylin and eosin, and levels of inflammation were scored as follows: 0, no inflammation; 1, presence of giant cells, occasional lymphocytes, and plasma cells; 2, presence of giant cells, plasma cells, eosinophils, and neutrophils; and 3, presence of many inflammatory cells and microabscesses. The level of fibrosis was scored as follows: 0, no fibrosis; 1, minimal, loose; 2, moderate; 3, florid dense [22]. 2.3. Immunohistochemistry Slides were immunostained with 100 mg of transforming growth factor beta-1 (TGF-b1) rat polyclonal antibody (Acris Antibodies GmbH; Schillerstraße 5, D-32052 Herford, Germany). The sections were incubated at 65 8C for 1 h overnight. Deparaffinization was performed using the following series of washes: two xylene washes (3 min each), two 100% ethanol rinses (3 min each), and one wash each of 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and Tris-buffered saline with 0.05% Tween (TBST; 3 min each wash on a shaker). For antigen retrieval, 500 mL of distilled water was added to a pressure cooker. The slides were immersed in a staining dish containing the antigen retrieval solution and then placed in a decloaking chamber. Sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) was used as the antigen retrieval buffer. The sections were boiled for approximately 10 min and cooled at room temperature. The slides were then removed and rinsed in TBST. For the staining procedure, the slides were washed with TBST and covered with 3% hydrogen peroxide to inactivate the endogenous peroxidase. The slides were washed three times with TBST for 3 min each on a shaker and blocked with a universal protein blocking solution for 1 h. The TGF-b1 rat antibody (AP06350PU-N) was diluted to 1:50. The primary antibody was applied to each section and incubated overnight in a humidified
Please cite this article in press as: Kaya C, et al. A randomized controlled study of the efficacy of misoprostol and hyaluronic acid in preventing adhesion formation after gynecological surgery: a rat uterine horn model. Eur J Obstet Gynecol (2014), http://dx.doi.org/ 10.1016/j.ejogrb.2014.02.033
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Table 1 Comparison of clinical, histopathological and immunohystochemistry scores of the control, misoprostol and Hyalobarrier1 groups. Parameters Number of uterine horns Clinical adhesion score Extent score Severity score Degree score Total score Histopathological score Inflammation Fibrosis Immunohystochemistry TGF b1 antibody
Control group
Misoprostol group
14 2.29 0.73 2.29 0.73 2.14 0.86 6.71 2.13
14 [2 [2 [2 [7
(1–3)] (1–3)] (1–3)] (3–9)]
0.86 0.86 0.86 0.86 0.86 0.86 2.57 2.59
Hyalobarrier1 group
p-Value
14 [1 [1 [1 [3
(0–2)] (0–2)] (0–2)] (0–6)]
0.86 0.86 0.86 0.86 0.86 0.86 2.57 2.47
[1 [1 [1 [3
(0–2)] (0–2)] (0–2)] (0–6)]
0.0001 0.0001 0.0001 0.0001
2 0 [2 (2–2)] 1.29 0.47 [1 (1–2)]
1.43 0.51 [1 (1–2)] 1.43 0.51 [1 (1–2)]
2.14 0.36 [2 (2–3)] 1.29 0.47 [1 (1–2)]
0.0001 0.658
1 0.78 [1 (0–2)]
0.71 0.47 [1 (0–1)]
0.86 0.66 [1 (0–2)]
0.581
TGF b1, transforming growth factor beta 1.
chamber at 4 8C. The slides were washed three times with TBST (3 min each on a shaker). Secondary HRP-conjugated antibody diluted in the blocking solution was applied to each section as per the manufacturer’s recommendations and incubated for 30 min at
room temperature. The slides were washed three times with TBST (5 min each on a shaker) and incubated with freshly prepared 3,30 diaminobenzidine substrate until the stain developed. The sections were rinsed with water and counterstained with hematoxylin.
Fig. 1. Clinical adhesion scoring. (a) No uterine adhesion, (b) 1–25% involvement, filmy avascular, the adhesion could be separated from tissue with gentle traction, (c) 26–50% involvement, vascular and opaque, the adhesion could be separated from tissue with moderate traction, and (d) 51–75% involvement, cohesive attachment of uterine horn to each other or other abdominal organs, requiring sharp dissection.
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Samples were then dehydrated by two washes with 100% ethanol (3 min each) and two rinses with xylene (3 min each). The slides were mounted with cover slips and all sections examined using a light microscope. Five randomly chosen fields of each horn were examined, and the number of positively stained cells among 100 cells was counted. A total of 500 cells were counted for each horn. The percentage of immunoreactive cells was recorded by an established scoring system as follows: 0, negative; 1, <33% positive staining; 2, 33–66% positive staining; 3, >66% positive staining [23].
Table 2 Comparison of p values for clinical adhesion scores and inflammation scores of the control, misoprostol and Hyalobarrier1 groups. Dunn test
Control group/ misoprostol group
Control group/ Hyalobarrier1 group
Control group/ Hyalobarrier1 group
Extent score Severity score Degree score Total score Inflammation
0.0001 0.0001 0.002 0.0001 0.001
0.0001 0.0001 0.002 0.0001 0.150
0.999 0.999 0.999 0.999 0.001
2.4. Statistical analysis Statistical data analysis was performed using the NCSS 2007 Statistical Software (Number Cruncher Statistical System; NCSS LLC, Kaysville, UT 84037, USA). Descriptive statistical methods (mean, standard deviation, min–max measurements) were used for data analysis. The Kruskal–Wallis test was used to compare differences in scores between the groups and the Dunn test was used to compare differences between subgroups. A p-value of <0.05 was considered statistically significant.
the Hyalobarrier1 group. The scores for the degree of adhesion formation were 2.14 0.86, 0.86 0.86, and 0.86 0.86 for the control, misoprostol, and Hyalobarrier1 groups, respectively. A statistically significant difference in these scores was noted between the misoprostol and control groups (p = 0.002) and the Hyalobarrier1 and control groups (p = 0.002), but there was no statistically significant difference in this parameter between the misoprostol and Hyalobarrier1 groups (Table 2 and Fig. 1A–D).
3. Results
3.2. Microscopic histological features
3.1. Macroscopic clinical adhesion scores
The median histological inflammation scores were 2 0, 1.43 0.51, and 2.14 0.36 for the control, misoprostol, and Hyalobarrier1 groups, respectively (Table 1). The inflammation score of the misoprostol group was significantly lower than that of the control and Hyalobarrier1 groups (p = 0.0001) (Table 2). However, there was no significant difference in the inflammation scores between the control and Hyalobarrier1 groups (Fig. 2A–D). There was also no significant difference in the fibrosis score and immunohistopathological scoring with TGF-b1 (Fig. 3A and B) between the study groups.
Total clinical adhesion formation scores were evaluated for the control, misoprostol, and Hyalobarrier1 groups, and the scores were 6.71 2.13, 2.57 2.59, and 2.57 2.47 respectively (Table 1). A statistically significant difference was found between the misoprostol and Hyalobarrier1 groups compared with the control group in the total adhesion scores (p = 0.0001), extent scores (p = 0.0001), and severity scores (p = 0.0001). However, there was no significant difference in these parameters between the misoprostol group and
Fig. 2. Histopathological inflammation evaluation of adhered tissues. Hematoxylin and eosin 200. (a) Giant cell formation, (b) microabscesses formation, (c) minimal, loose fibrosis, and (d) moderate fibrosis.
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Fig. 3. Histopathological TGF b1 antibody evaluation of adhered tissues. (a) TGF b1 antibody 400, 1–33% involvement and (b) TGF b1 antibody 400, 34–66% involvement.
4. Comment Development of postoperative adhesions remains a major healthcare problem causing relaparotomy complications, extended operation time, additional blood loss, and unexpected visceral damage [24]. Adhesions occur after damage to the mesothelial cells on the serosal surface of tissues, followed by inflammation with the release of cytokines, and the formation of a soft fibrin gel matrix. If this soft fibrin is not fractioned and fibrinolytic activity is insufficient, dense fibrinous and vascular changes develop within two weeks [5]. To minimize this undesirable fibrin formation, several approaches have been suggested. These include minimizing the tissue injury, removing the fibrin, and inhibiting the proliferation of fibroblasts at the injured site [10]. A number of agents have been studied for this purpose, such as fibrinolytic agents to reduce the fibrinous mass, nonsteroidal anti-inflammatory drugs, corticosteroids, statins, metformin, methylene blue, cyclooxygenase-2 inhibitors, PPARc and VEGF receptor inhibitors, anti-oxidants, aromatase inhibitors, GnRH-agonists, and human amniotic membrane. There is, however, no optimal method for adhesion prevention [5,10,20,25–29]. Hyaluronan (or hyaluronic acid) derivatives prevent adhesions by reducing fibrin formation and promoting the production of peritoneal mesothelial cells [11]. Hyalobarrier1 is a reported adhesion-preventing agent [15]. Furthermore, a systematic review and meta-analysis reported that the application of the autocrosslinked hyaluronan gel significantly reduced the incidence of intraperitoneal adhesions after laparoscopic myomectomy and incidence of intrauterine adhesions after hysteroscopy [11]. Hyalobarrier1 gel is also useful in its simple, ready-to-use form. In a multicenter study, Hyalobarrier1 did not significantly prolong operating time, and the mean application time of the product was two minutes [30]. In our institution, Hyalobarrier1 gel is used as an adhesion-preventing agent in routine surgical procedures. Misoprostol, a drug that is commonly used by obstetricians for the induction of labor and facilitation of cervical dilation before intrauterine procedures, alters the microstructural architecture and enzymatic processes of collagen [16]. In our study, we showed that both misoprostol and Hyalobarrier1 significantly reduced the clinical adhesion score compared with that of the control group. To the best of our knowledge, this is the first study evaluating the effect of misoprostol on adhesion prevention. Fibrin formation following the inflammatory process is believed to be the major factor in the development of surgical adhesions. This inflammatory process begins after tissue damage and lasts throughout collagen fiber synthesis that enhances scar formation [10]. Although the mechanism of action of misoprostol in reducing collagen fiber formation has not been completely described, we found that the histopathological inflammation score of the misoprostol group was significantly lower than that of the Hyalobarrier1 and control
groups. We can speculate that misoprostol may prevent adhesion by reducing the action of inflammatory cytokines involved in the first step of fibrin formation. In our study, we also performed fibrosis scoring and TGF-b1 rat antibody immunohistochemistry scoring. TGF-b1 has a welldescribed role in cell differentiation, in triggering angiogenesis, and as a fibroblast activator in the inflammatory response [31]. However, there was no significant difference found in the TGF-b1 antibody immunohistochemistry scores between the control and treatment groups. We postulate that, as a limitation of our study, the recovery period following the initial surgery was 14 days, which is the minimum stabilization time limit for collagen synthesis. Collagen band formation typically begins 3–5 days following trauma and increases until stabilization at approximately day 14 [32]. Therefore, assessments should be performed after 14 days to provide more accurate immunohistochemistry results. Most adhesion-preventing agents are administered locally in daily surgical practice [30]. In our study, the preferred route of administration was IP because surgeons generally prefer applying agents via small instruments to prevent adhesion during minimally invasive surgical techniques. We used 10 mcg/kg of a Cytotec1 tablet, as this was the described minimal dose of intraperitoneal misoprostol to produce its gastroprotective effect [33]. The limitations of our study are that the required dose of misoprostol was obtained by dissolving a Cytotec1 200 mcg tablet in distilled water and that the effects of the tablet components on adhesion prevention or formation are unknown. The optimal dose and administration route of misoprostol are also unknown; therefore, pharmacokinetic studies should be performed on the use of misoprostol in preventing pelvic adhesions. Moreover, the cellular and molecular interactions of misoprostol in this situation need to be investigated further. Conflicts of interest statement The authors have no conflicts of interest to declare. Acknowledgements We are grateful to thank to Ays¸enur Turan for her technical support and to Rana Konyalioglu for statistical analyses of the data. References [1] Hellebrekers BWJ, Trimbos-Kemper GCM, Blitterswijk VCA, Bakkum EA, Trimbos JBMZ. Effects of five different barrier materials on postsurgical adhesion formation in the rat. Hum Reprod 2000;15:1358–63. [2] Aritas¸ Y, Akcan A, Erdogan AR, Akgu¨n H, Saraymen R, Akyildiz H. Effects of melatonin and phospholipid on adhesion formation and correlation with
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A randomized controlled study of the efficacy of misoprostol and hyaluronic acid in preventing adhesion formation after gynecological surgery: a rat uterine horn model Cihan Kaya a,*, Nurten Sever b, Hu¨seyin Cengiz c, S¸u¨kru¨ Yıldız c, Murat Ekin c, Levent Yas¸ar c Van O¨zalp State Hospital, Clinic of Obstetrics and Gynecology, Turkey Bakirkoy Dr Sadi Konuk Training and Research Hospital, Department of Pathology, Turkey c Bakirkoy Dr Sadi Konuk Training and Research Hospital, Department of Obstetrics and Gynecology, Turkey a
b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 December 2013 Received in revised form 3 February 2014 Accepted 14 February 2014
Objective: To investigate the effect of misoprostol in the reduction of adhesion formation after gynecological surgery. Study design: A double blind, randomized controlled experimental study was designed. Twenty-one female Wistar Hannover rats were divided into three groups as control, misoprostol and Hyalobarrier1 groups. A uterine horn adhesion model was created. After anesthesia induction, 1.5–2 cm injuries were made to the each uterine horn by cautery. The control group received no special medications except for the standard surgical procedure. The misoprostol group received 10 mcg/kg misoprostol in addition to the standard surgical procedure, and the Hyalobarrier1 group received 1 cm3 ready-for-use Hyalobarrier1 gel intraperitoneally in addition to the standard surgical procedure. After 14 days from the first surgical procedure, adhesion scores were evaluated. Results: The extent (p < 0.001), severity (p < 0.001), degree (p < 0.001) and total adhesion score (p < 0.001) values of the control group were statistically higher than the values of misoprostol and Hyalobarrier1 groups. The inflammation score value of misoprostol group was statistically lower than control and Hyalobarrier1 groups (p < 0.001). Conclusion: In this study, we have found a new therapeutic potential of misoprostol that may be useful in preventing pelvic adhesion and reducing inflammation scores. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Surgical adhesions Hyaluronic acid Misoprostol PGE1 TGF b1
1. Introduction Postoperative intra-abdominal adhesions occur in 50–95% of women who undergo gynecological surgery [1]. The development of peritoneal adhesions following abdominal and pelvic surgery leads to clinical problems, including intestinal obstruction, infertility, and chronic pelvic pain [2]. Adhesions remain a potential problem for future surgical procedures, increasing health expenses and hospital readmission rates, and reducing the quality of life of the patient [3]. There are several described causes of adhesions, such as mechanical trauma, ischemia caused by sutures or electrocautery, presence of foreign bodies, tissue desiccation,
¨ zalp State Hospital, Clinic of Obstetrics and * Corresponding author at: Van O ¨ zalp, Van, Turkey. Gynecology, Cumhuriyet Mh., No. 3 Merkez O Tel.: +90 212 414 73 72. E-mail address: [email protected] (C. Kaya).
and infection [4]. Furthermore, during peritoneal healing following surgery, the increase in peritoneal fluid, cytokines, proteins and fibrin formation can promote adhesion formation [5,6]. Currently, there are various methods of preventing adhesion formation such as the use of barrier materials, non-steroid antiinflammatory drugs, calcium channel blockers, corticosteroids, vitamin E, antihistamines, metformin, melatonin, progesterone, estrogen, gonadotrophin-releasing hormone (GnRH)-agonists and antagonists, anticoagulants, fibrinolytic agents, and antibiotics [7– 10]. Hyalobarrier1 gel is a highly viscous gel derived from hyaluronan, obtained through an auto-crosslinking process, and used as an adhesion prevention agent in a variety of surgical procedures [11–13]. Hyalobarrier1 gel can reduce the incidence and severity of postoperative adhesions in cases of severe uterine damage caused by laparotomy or laparoscopic myomectomies [14,15]. The gel can also be easily applied during laparoscopic and hysteroscopic procedures.
http://dx.doi.org/10.1016/j.ejogrb.2014.02.033 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Kaya C, et al. A randomized controlled study of the efficacy of misoprostol and hyaluronic acid in preventing adhesion formation after gynecological surgery: a rat uterine horn model. Eur J Obstet Gynecol (2014), http://dx.doi.org/ 10.1016/j.ejogrb.2014.02.033
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Misoprostol, a synthetic analog of prostaglandin E1, is used as an anti-ulcer drug that increases gastric mucus secretion and reduces gastric acid secretion [16]. Misoprostol is also used offlabel by obstetricians for the induction of first trimester abortion [17]. Misoprostol has been shown to alter enzymatic processes that cause degradation and remodeling of collagen fibers [18]. Nevertheless, no in vivo studies have demonstrated that misoprostol can reduce adhesion formation. Misoprostol is commercially available as Cytotec1 (Ali Raif I˙lac¸, I˙stanbul, Tu¨rkiye) in a pharmaceutical complex with hydroxypropyl methylcellulose, microcrystalline cellulose, sodium starch glycolate, and hydrogenated castor oil [19]. In this study, we aimed to investigate the effect of the intraperitoneal use of misoprostol in the prevention of adhesions by reducing collagen synthesis, which plays a major role in wound healing and scar formation, compared with that of Hyalobarrier1 gel, which is known to prevent adhesions in clinical practice. 2. Materials and methods The study was approved by the local ethical review board of the Bagcilar Training and Research Hospital in Istanbul, Turkey, for the use of laboratory animals, and was performed at the experimental surgery training and education center of the same hospital (approval number: 2013–17). 2.1. Animal maintenance and treatment Rats weighing approximately 180–260 g were housed under the institutional review board’s guidelines for animal care using a day cycle of 14 h of light with free access to food and water. A power analysis was performed to calculate the minimum sample size required for the study (alpha error = 0.05 and 1-beta = 0.8) and at least 12 uterine horns were required for each study group. As 10% of the animals are lost during the procedures, we included in total 14 horns. The rats were randomly assigned to three study groups, each consisting of seven rats, using computer-based randomization. Twenty-one mature, nonpregnant, female Wistar Hannover rats (aged 8–10 weeks) were used as models for the experimental induction of postoperative uterine horn adhesion, as defined by Kelekci et al. [20]. Animals were anesthetized using 50 mg/kg of 10% ketamine hydrochloride (Ketasol; Richter Pharma AG, Wels, Austria) intramuscularly (IM) and 5 mg/kg of 2% xylazine (Rompun; Bayer Health Care LCC, Kansas, KS) IM. Prior to the operation, the abdominal skin was shaved and disinfected using 10% povidone-iodine solution (Batticon; Adeka Laboratories, Istanbul, Turkey). A 3-cm midline incision was made, and the uterine horns were exposed. Seven standard lesions were inflicted in the antimesenteric surfaces of each uterine horn using a 10 W bipolar cautery (Bovie1, FL, USA). An area of 1.5 cm 1.5 cm between the two main vascular branches of the lower side wall at the level of each uterine horn was cauterized to produce an opposing lesion. The first group of rats served as the control group and received no special medication. The second group (misoprostol group) received 10 mcg/kg misoprostol intraperitoneally (IP) using a diluted Cytotec1 200 mcg tablet (Ali Raif I˙lac¸, I˙stanbul, Tu¨rkiye). The third group (Hyalobarrier1 gel group) received 1 mL of readyto-use Hyalobarrier1 gel (Fidia Advanced Biopolymers S.r.I Abano Terme, Italy) IP. The abdominal incision was closed in two layers with a 4-0 polyglycolic acid suture (VicrylTM; Johnson and Johnson Medical Limited, Ethicon Limited, UK) for the peritoneum and 3-0 polyglactin suture for the skin. After the animals recovered from surgery, they were housed separately under controlled temperatures of 22 2 8C and a 14 h light cycle with food and water ad libitum. The surgery was limited to approximately 15 min for each rat to prevent tissue drying at room
temperature. All the surgical procedures were performed by the same researchers. 2.2. Tissue sample collection and histopathology Following the 14-day recovery period, the rats were sacrificed by cervical dislocation. The adhesion areas were evaluated and graded macroscopically by two observers who were blinded to the study groups. The extent and severity of the adhesions were evaluated using an established scoring system [21]. The extent of adhesion spread to the uterine horn defect was scored as follows: 0, no uterine adhesion; 1, 1–25% involvement, 2, 26–50% involvement, 3, 51–75% involvement, and 4, 76–100% involvement. Adhesions were further characterized on gross examination according to the severity of attachment between the uterine horns and other abdominal organs as follows: 0, no adhesion, 1, filmy avascular, 2, vascular or opaque, and 3, cohesive. The degree of adhesion formation was evaluated and scored using the following; 0, no adhesion; 1, the adhesion could be separated from the tissue with gentle traction, 2, the adhesion could be separated from the tissue with moderate traction, 3, the adhesion could be separated from the tissue by sharp dissection. The sum of the three parameters was used as the total score for each group. Tissue samples were obtained from all serosal surfaces where adhesions had developed. One histologist evaluated all tissues and was blinded to the origin of the samples. Tissues were graded for levels of inflammation and fibrosis using previously published grading scales. For histological analyses, the excised tissues were fixed in 10% buffered formalin solution for 24 h. After fixation, a routine tissue-processing procedure was performed, and sample tissues were embedded in paraffin. Paraffin wax blocks were cut into 4-mm thick sections. Prepared sections were then stained with hematoxylin and eosin, and levels of inflammation were scored as follows: 0, no inflammation; 1, presence of giant cells, occasional lymphocytes, and plasma cells; 2, presence of giant cells, plasma cells, eosinophils, and neutrophils; and 3, presence of many inflammatory cells and microabscesses. The level of fibrosis was scored as follows: 0, no fibrosis; 1, minimal, loose; 2, moderate; 3, florid dense [22]. 2.3. Immunohistochemistry Slides were immunostained with 100 mg of transforming growth factor beta-1 (TGF-b1) rat polyclonal antibody (Acris Antibodies GmbH; Schillerstraße 5, D-32052 Herford, Germany). The sections were incubated at 65 8C for 1 h overnight. Deparaffinization was performed using the following series of washes: two xylene washes (3 min each), two 100% ethanol rinses (3 min each), and one wash each of 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, and Tris-buffered saline with 0.05% Tween (TBST; 3 min each wash on a shaker). For antigen retrieval, 500 mL of distilled water was added to a pressure cooker. The slides were immersed in a staining dish containing the antigen retrieval solution and then placed in a decloaking chamber. Sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0) was used as the antigen retrieval buffer. The sections were boiled for approximately 10 min and cooled at room temperature. The slides were then removed and rinsed in TBST. For the staining procedure, the slides were washed with TBST and covered with 3% hydrogen peroxide to inactivate the endogenous peroxidase. The slides were washed three times with TBST for 3 min each on a shaker and blocked with a universal protein blocking solution for 1 h. The TGF-b1 rat antibody (AP06350PU-N) was diluted to 1:50. The primary antibody was applied to each section and incubated overnight in a humidified
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Table 1 Comparison of clinical, histopathological and immunohystochemistry scores of the control, misoprostol and Hyalobarrier1 groups. Parameters Number of uterine horns Clinical adhesion score Extent score Severity score Degree score Total score Histopathological score Inflammation Fibrosis Immunohystochemistry TGF b1 antibody
Control group
Misoprostol group
14 2.29 0.73 2.29 0.73 2.14 0.86 6.71 2.13
14 [2 [2 [2 [7
(1–3)] (1–3)] (1–3)] (3–9)]
0.86 0.86 0.86 0.86 0.86 0.86 2.57 2.59
Hyalobarrier1 group
p-Value
14 [1 [1 [1 [3
(0–2)] (0–2)] (0–2)] (0–6)]
0.86 0.86 0.86 0.86 0.86 0.86 2.57 2.47
[1 [1 [1 [3
(0–2)] (0–2)] (0–2)] (0–6)]
0.0001 0.0001 0.0001 0.0001
2 0 [2 (2–2)] 1.29 0.47 [1 (1–2)]
1.43 0.51 [1 (1–2)] 1.43 0.51 [1 (1–2)]
2.14 0.36 [2 (2–3)] 1.29 0.47 [1 (1–2)]
0.0001 0.658
1 0.78 [1 (0–2)]
0.71 0.47 [1 (0–1)]
0.86 0.66 [1 (0–2)]
0.581
TGF b1, transforming growth factor beta 1.
chamber at 4 8C. The slides were washed three times with TBST (3 min each on a shaker). Secondary HRP-conjugated antibody diluted in the blocking solution was applied to each section as per the manufacturer’s recommendations and incubated for 30 min at
room temperature. The slides were washed three times with TBST (5 min each on a shaker) and incubated with freshly prepared 3,30 diaminobenzidine substrate until the stain developed. The sections were rinsed with water and counterstained with hematoxylin.
Fig. 1. Clinical adhesion scoring. (a) No uterine adhesion, (b) 1–25% involvement, filmy avascular, the adhesion could be separated from tissue with gentle traction, (c) 26–50% involvement, vascular and opaque, the adhesion could be separated from tissue with moderate traction, and (d) 51–75% involvement, cohesive attachment of uterine horn to each other or other abdominal organs, requiring sharp dissection.
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Samples were then dehydrated by two washes with 100% ethanol (3 min each) and two rinses with xylene (3 min each). The slides were mounted with cover slips and all sections examined using a light microscope. Five randomly chosen fields of each horn were examined, and the number of positively stained cells among 100 cells was counted. A total of 500 cells were counted for each horn. The percentage of immunoreactive cells was recorded by an established scoring system as follows: 0, negative; 1, <33% positive staining; 2, 33–66% positive staining; 3, >66% positive staining [23].
Table 2 Comparison of p values for clinical adhesion scores and inflammation scores of the control, misoprostol and Hyalobarrier1 groups. Dunn test
Control group/ misoprostol group
Control group/ Hyalobarrier1 group
Control group/ Hyalobarrier1 group
Extent score Severity score Degree score Total score Inflammation
0.0001 0.0001 0.002 0.0001 0.001
0.0001 0.0001 0.002 0.0001 0.150
0.999 0.999 0.999 0.999 0.001
2.4. Statistical analysis Statistical data analysis was performed using the NCSS 2007 Statistical Software (Number Cruncher Statistical System; NCSS LLC, Kaysville, UT 84037, USA). Descriptive statistical methods (mean, standard deviation, min–max measurements) were used for data analysis. The Kruskal–Wallis test was used to compare differences in scores between the groups and the Dunn test was used to compare differences between subgroups. A p-value of <0.05 was considered statistically significant.
the Hyalobarrier1 group. The scores for the degree of adhesion formation were 2.14 0.86, 0.86 0.86, and 0.86 0.86 for the control, misoprostol, and Hyalobarrier1 groups, respectively. A statistically significant difference in these scores was noted between the misoprostol and control groups (p = 0.002) and the Hyalobarrier1 and control groups (p = 0.002), but there was no statistically significant difference in this parameter between the misoprostol and Hyalobarrier1 groups (Table 2 and Fig. 1A–D).
3. Results
3.2. Microscopic histological features
3.1. Macroscopic clinical adhesion scores
The median histological inflammation scores were 2 0, 1.43 0.51, and 2.14 0.36 for the control, misoprostol, and Hyalobarrier1 groups, respectively (Table 1). The inflammation score of the misoprostol group was significantly lower than that of the control and Hyalobarrier1 groups (p = 0.0001) (Table 2). However, there was no significant difference in the inflammation scores between the control and Hyalobarrier1 groups (Fig. 2A–D). There was also no significant difference in the fibrosis score and immunohistopathological scoring with TGF-b1 (Fig. 3A and B) between the study groups.
Total clinical adhesion formation scores were evaluated for the control, misoprostol, and Hyalobarrier1 groups, and the scores were 6.71 2.13, 2.57 2.59, and 2.57 2.47 respectively (Table 1). A statistically significant difference was found between the misoprostol and Hyalobarrier1 groups compared with the control group in the total adhesion scores (p = 0.0001), extent scores (p = 0.0001), and severity scores (p = 0.0001). However, there was no significant difference in these parameters between the misoprostol group and
Fig. 2. Histopathological inflammation evaluation of adhered tissues. Hematoxylin and eosin 200. (a) Giant cell formation, (b) microabscesses formation, (c) minimal, loose fibrosis, and (d) moderate fibrosis.
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Fig. 3. Histopathological TGF b1 antibody evaluation of adhered tissues. (a) TGF b1 antibody 400, 1–33% involvement and (b) TGF b1 antibody 400, 34–66% involvement.
4. Comment Development of postoperative adhesions remains a major healthcare problem causing relaparotomy complications, extended operation time, additional blood loss, and unexpected visceral damage [24]. Adhesions occur after damage to the mesothelial cells on the serosal surface of tissues, followed by inflammation with the release of cytokines, and the formation of a soft fibrin gel matrix. If this soft fibrin is not fractioned and fibrinolytic activity is insufficient, dense fibrinous and vascular changes develop within two weeks [5]. To minimize this undesirable fibrin formation, several approaches have been suggested. These include minimizing the tissue injury, removing the fibrin, and inhibiting the proliferation of fibroblasts at the injured site [10]. A number of agents have been studied for this purpose, such as fibrinolytic agents to reduce the fibrinous mass, nonsteroidal anti-inflammatory drugs, corticosteroids, statins, metformin, methylene blue, cyclooxygenase-2 inhibitors, PPARc and VEGF receptor inhibitors, anti-oxidants, aromatase inhibitors, GnRH-agonists, and human amniotic membrane. There is, however, no optimal method for adhesion prevention [5,10,20,25–29]. Hyaluronan (or hyaluronic acid) derivatives prevent adhesions by reducing fibrin formation and promoting the production of peritoneal mesothelial cells [11]. Hyalobarrier1 is a reported adhesion-preventing agent [15]. Furthermore, a systematic review and meta-analysis reported that the application of the autocrosslinked hyaluronan gel significantly reduced the incidence of intraperitoneal adhesions after laparoscopic myomectomy and incidence of intrauterine adhesions after hysteroscopy [11]. Hyalobarrier1 gel is also useful in its simple, ready-to-use form. In a multicenter study, Hyalobarrier1 did not significantly prolong operating time, and the mean application time of the product was two minutes [30]. In our institution, Hyalobarrier1 gel is used as an adhesion-preventing agent in routine surgical procedures. Misoprostol, a drug that is commonly used by obstetricians for the induction of labor and facilitation of cervical dilation before intrauterine procedures, alters the microstructural architecture and enzymatic processes of collagen [16]. In our study, we showed that both misoprostol and Hyalobarrier1 significantly reduced the clinical adhesion score compared with that of the control group. To the best of our knowledge, this is the first study evaluating the effect of misoprostol on adhesion prevention. Fibrin formation following the inflammatory process is believed to be the major factor in the development of surgical adhesions. This inflammatory process begins after tissue damage and lasts throughout collagen fiber synthesis that enhances scar formation [10]. Although the mechanism of action of misoprostol in reducing collagen fiber formation has not been completely described, we found that the histopathological inflammation score of the misoprostol group was significantly lower than that of the Hyalobarrier1 and control
groups. We can speculate that misoprostol may prevent adhesion by reducing the action of inflammatory cytokines involved in the first step of fibrin formation. In our study, we also performed fibrosis scoring and TGF-b1 rat antibody immunohistochemistry scoring. TGF-b1 has a welldescribed role in cell differentiation, in triggering angiogenesis, and as a fibroblast activator in the inflammatory response [31]. However, there was no significant difference found in the TGF-b1 antibody immunohistochemistry scores between the control and treatment groups. We postulate that, as a limitation of our study, the recovery period following the initial surgery was 14 days, which is the minimum stabilization time limit for collagen synthesis. Collagen band formation typically begins 3–5 days following trauma and increases until stabilization at approximately day 14 [32]. Therefore, assessments should be performed after 14 days to provide more accurate immunohistochemistry results. Most adhesion-preventing agents are administered locally in daily surgical practice [30]. In our study, the preferred route of administration was IP because surgeons generally prefer applying agents via small instruments to prevent adhesion during minimally invasive surgical techniques. We used 10 mcg/kg of a Cytotec1 tablet, as this was the described minimal dose of intraperitoneal misoprostol to produce its gastroprotective effect [33]. The limitations of our study are that the required dose of misoprostol was obtained by dissolving a Cytotec1 200 mcg tablet in distilled water and that the effects of the tablet components on adhesion prevention or formation are unknown. The optimal dose and administration route of misoprostol are also unknown; therefore, pharmacokinetic studies should be performed on the use of misoprostol in preventing pelvic adhesions. Moreover, the cellular and molecular interactions of misoprostol in this situation need to be investigated further. Conflicts of interest statement The authors have no conflicts of interest to declare. Acknowledgements We are grateful to thank to Ays¸enur Turan for her technical support and to Rana Konyalioglu for statistical analyses of the data. References [1] Hellebrekers BWJ, Trimbos-Kemper GCM, Blitterswijk VCA, Bakkum EA, Trimbos JBMZ. Effects of five different barrier materials on postsurgical adhesion formation in the rat. Hum Reprod 2000;15:1358–63. [2] Aritas¸ Y, Akcan A, Erdogan AR, Akgu¨n H, Saraymen R, Akyildiz H. Effects of melatonin and phospholipid on adhesion formation and correlation with
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