European Journal of Obstetrics & Gynecology and Reproductive Biology 177 (2014) 44–47
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effect of intraabdominal administration of Allium sativum (garlic) oil on postoperative peritoneal adhesion Ahmet Sahbaz a,*, Hatice Isik a, Oner Aynioglu a, Kemal Gungorduk b, Banu Dogan Gun c a
Zonguldak Bulent Ecevit University, School of Medicine, Department of Obstetrics and Gynecology, Kozlu, Zonguldak, Turkey Izmir Tepecik Education and Research Hospital, Department of Gynaecologic Oncology, Izmir, Turkey c Zonguldak Bulent Ecevit University, School of Medicine, Department of Pathology, Kozlu, Zonguldak, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 October 2013 Received in revised form 22 March 2014 Accepted 25 March 2014
Objective(s): Peritoneal adhesion is a serious problem that develops after most abdominopelvic surgeries. Allium sativum (garlic) has been used for centuries as both a nutrient and a traditional medicine. The anti-inflammatory, antibacterial, fibrinolytic, and wound-healing properties of garlic are well-recognized. We evaluated the effect of garlic on formation of postoperative adhesions in rats. Study design: Thirty Wistar-Albino female rats weighing 200–250 g were randomly divided into three groups (10 rats/group). Group 1 rats received 5 ml/kg garlic oil intraperitoneally and no surgery was performed. The ceca of Group 2 rats (controls) were scraped, to trigger adhesion formation, and no treatment was given. In Group 3 rats, 5 ml/kg garlic oil was added to the peritoneal cavity immediately after the cecum was scraped. All animals were sacrificed 10 d after surgery and adhesions graded in terms of severity and histopathologic characteristics. Results: All animals tolerated the operations well. No adhesions were evident upon laparotomy of Group 1 animals. In Group 2 three rats had an adhesion grade 2 and seven rats had an adhesion of grade 3, whereas in Group 3 no adhesions were found in four rats, five rats had an adhesion grade of 1. Only one rat had a grade 2 adhesion. Macroscopic adhesions and mean adhesion scores of Group 3 were significantly lower than Group 2 (p < 0.001). Histopathologic evaluation of the specimens also revealed a statistically significant differences in inflammation, fibrosis, and neovascularization scores between Group 2 and 3 (p = 0.001, p = 0.001, and p = 0.011, respectively). Inflammation, fibrosis and vascularization scores in Group 3 were found significantly lower than Group 2. Conclusion: The anti-inflammatory, antibacterial, fibrinolytic, antithrombotic, and wound-healing effects of garlic likely prevent formation of peritoneal adhesions in a rat model, and garlic may be effective and cheap when used to prevent such adhesions in humans. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adhesion Allium sativum Garlic Peritoneal Prevention Postoperative
1. Introduction Postoperative peritoneal adhesion (PPA), defined as development of fibrous bands between peritoneal surfaces, is a serious and common (90% of patients) complication of abdominopelvic surgery [1,2]. PPA is triggered by disruption of the normal healing process. Infection, ischemia, hemorrhage, trauma, and foreign body reaction are the principal causes of adhesion formation after surgery [2]. PPA causes various health problems, including bowel obstruction and chronic pelvic pain, and is also a leading cause of female
* Corresponding author. Tel.: +90 5054439900. E-mail address: [email protected] (A. Sahbaz). http://dx.doi.org/10.1016/j.ejogrb.2014.03.018 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
infertility [2–4]. PPA is devastating to millions and a major financial burden on healthcare systems. Worldwide, the total annual cost of hospitalization (including surgery) of PPA patients is about $1.3 billion [5]. Anti-inflammatory agents, antioxidants, anticoagulants, and fibrinolytics have been used in efforts to prevent adhesion [6], but no optimal treatment option has yet been identified. Allium sativum (garlic), of the Liliaceae, has been used as both a nutrient and a folk medicine for centuries. Sulfur-containing compounds, including diallyl trisulfide (DAT) and diallyl sulfide (DAD) give garlic its well-known flavor, and exert physiological effects [7,8]. The antibacterial, anti-inflammatory, antithrombotic, fibrinolytic, antioxidant, and wound-healing properties of garlic have been well-documented in previous studies [7–9]. We thus hypothesized that garlic oil might prevent PPA.
A. Sahbaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 177 (2014) 44–47
45
2. Materials and methods
2.1. Adhesion grading
This study was approved by the Ethics Committee for Animal Experiments of Zonguldak Bulent Ecevit University and was performed in the Experimental Animal Production and Research Laboratory of the Medical School of that University, Zonguldak, Turkey. Thirty female Wistar rats (200–250 g, caged in groups of five) were used. Rats were fed ad libitum with standard pelleted food approved by the Turkish Standards Institute and had free access to tap water. The rats were randomly divided into three groups of 10 rats each. Group 1 rats received 5 ml/kg garlic oil (Oleum Allii SativiR; Arifoglu, Firuzkoy Esenyurt Yolu No. 32 Avcilar, I˙stanbul) via injection into the peritoneal cavity using 22-French syringes (Fig. 1A). The dose of garlic oil was determined as suggested by Chen et al. [10]. Rats were anesthesized by intramuscular injection of ketamine (75 mg/kg; Ketalar; Parke-Davis and Co. Inc., Detroit, MI) and 40 mg/kg xylazine (Rompun; Bayer, Berlin, Germany). Each rat was placed on its back on an operating table. The abdominal skin was sterilized with povidone iodine (Betadine; Kurtsan Co., Istanbul, Turkey) and a vertical midline incision (3 cm long) was made. The terminal ileum and cecum were mobilized and draped over the left index finger of the surgeon. The anterior surface of each cecum was scraped with dry gauze to induce serosal petechiae (Fig. 1B) and arteries supplying the scraped segment were clamped for 1 min to induce transient ischemia (the ‘‘scraping model’’) [11]. The cecum was replaced and the incision closed with continuous 3-0 polypropylene sutures (Prolene; Bicakcilar Co., Istanbul, Turkey). Group 2 (control) rats received no treatment. Group 3 (test) rats were treated with 5 ml/kg garlic oil, delivered into the peritoneal cavity immediately after scraping. All rats were next individually caged, weighed regularly, and observed closely for development of surgical complications. Ten days later, all animals were sacrificed via intraperitoneal injection of a high dose of sodium pentothal. Reverse-U incisions were made to allow of maximal exploration.
The modified Evans adhesion scoring system, which is simple and logical, was used [12]. In this system, grade 0 = no adhesions; grade 1 = smooth adhesions that split either spontaneously or upon application of weak traction; grade 2 = firm adhesions splitting when traction is applied; and grade 3 = dense adhesions requiring dissection with a blade [12]. The same researcher, blinded to Group, determined all adhesion scores. The strongest adhesion identified in a rat defined the adhesion grade of that rat (Fig. 2).
[(Fig._1)TD$IG]
Fig. 1. (A) Intraperitoneal administration of garlic oil. (B) Serosal petechiae.
2.2. Histopathologic evaluation The cecal area and associated adhesive lesions were resected and preserved in formalin solution. After routine histological examination process, 5 mm thick sections were prepared and stained with hematoxylin and eosin. Under a light microscope, the histological sections were examined and scored for the presence of inflammatory cell infiltrate (0: absent or normal in number, 1: slight increase, 2: moderate infiltration, 3: massive infiltration), fibrosis (0: none, 1: slight, 2: moderate, 3: dense) and vascularization (0: none, 1: one to two vessels, 2: three to nine vessels, 3: ten or more vessels) (Fig. 3). Statistical analysis was performed using the MedCalc software (version 11.5 for Windows). The nonparametric Mann–Whitney Utest was used to explore the significance of among-Group differences in the grading of adhesions. A p value < 0.05 was considered ‘‘significant’’ and p < 0.001 ‘‘highly significant’’.
3. Results All animals tolerated the operations well. No signs of infection were observed in the incision site throughout the study period. No adhesions were evident upon laparotomy of Group 1 animals (no operation, but intraperitoneal admistration of garlic oil to evaluate any adhesion formation related to garlic oil use). In Group 2 three rats had an adhesion grade 2 and seven rats had an adhesion of grade 3, whereas in Group 3 no adhesions were found in four rats,
[(Fig._2)TD$IG]
Fig. 2. Macroscopic scores of adhesions. (A) Grade 0. (B) Grade 1. (C) Grade 2. (D) Grade 3.
[(Fig._3)TD$IG]
A. Sahbaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 177 (2014) 44–47
46
neovascularization scores between Group 2 and 3 (p = 0.001, p = 0.001, p = 0.011 respectively). Inflammation, fibrosis and vascularization scores in Group 3 were found significantly lower than Group 2. 4. Comments
Fig. 3. (a) Grade 3 inflammation with giant cell formation (H&E, 40). (b) Capillary vessel proliferation (grade 2 neovascularization) (H&E, 100). (c) Moderate fibrosis (grade 2) represented by young fibroblasts (H&E, 200).
five rats had an adhesion grade of 1. Only one rat had a grade 2 adhesion. Macroscopic adhesions and mean adhesions scores of the groups are summarized in Table 1. The adhesion scores of Group 3 were significantly lower compared to Group 2 (p < 0.001). Histopathologic evaluation scores of the groups with respect to inflammation, fibrosis and neovascularization are shown in Table 1. Histopathologic evaluation of the specimens also revealed a statistically significant differences in inflammation, fibrosis, and Table 1 Mean adhesion, inflammation, fibrosis, neovascularization scores. Group 2 n = 10
Group 3 n = 10
p value
2.70 0.48
0.70 0.67
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Effect of intraabdominal administration of Allium sativum (garlic) oil on postoperative peritoneal adhesion Ahmet Sahbaz a,*, Hatice Isik a, Oner Aynioglu a, Kemal Gungorduk b, Banu Dogan Gun c a
Zonguldak Bulent Ecevit University, School of Medicine, Department of Obstetrics and Gynecology, Kozlu, Zonguldak, Turkey Izmir Tepecik Education and Research Hospital, Department of Gynaecologic Oncology, Izmir, Turkey c Zonguldak Bulent Ecevit University, School of Medicine, Department of Pathology, Kozlu, Zonguldak, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 October 2013 Received in revised form 22 March 2014 Accepted 25 March 2014
Objective(s): Peritoneal adhesion is a serious problem that develops after most abdominopelvic surgeries. Allium sativum (garlic) has been used for centuries as both a nutrient and a traditional medicine. The anti-inflammatory, antibacterial, fibrinolytic, and wound-healing properties of garlic are well-recognized. We evaluated the effect of garlic on formation of postoperative adhesions in rats. Study design: Thirty Wistar-Albino female rats weighing 200–250 g were randomly divided into three groups (10 rats/group). Group 1 rats received 5 ml/kg garlic oil intraperitoneally and no surgery was performed. The ceca of Group 2 rats (controls) were scraped, to trigger adhesion formation, and no treatment was given. In Group 3 rats, 5 ml/kg garlic oil was added to the peritoneal cavity immediately after the cecum was scraped. All animals were sacrificed 10 d after surgery and adhesions graded in terms of severity and histopathologic characteristics. Results: All animals tolerated the operations well. No adhesions were evident upon laparotomy of Group 1 animals. In Group 2 three rats had an adhesion grade 2 and seven rats had an adhesion of grade 3, whereas in Group 3 no adhesions were found in four rats, five rats had an adhesion grade of 1. Only one rat had a grade 2 adhesion. Macroscopic adhesions and mean adhesion scores of Group 3 were significantly lower than Group 2 (p < 0.001). Histopathologic evaluation of the specimens also revealed a statistically significant differences in inflammation, fibrosis, and neovascularization scores between Group 2 and 3 (p = 0.001, p = 0.001, and p = 0.011, respectively). Inflammation, fibrosis and vascularization scores in Group 3 were found significantly lower than Group 2. Conclusion: The anti-inflammatory, antibacterial, fibrinolytic, antithrombotic, and wound-healing effects of garlic likely prevent formation of peritoneal adhesions in a rat model, and garlic may be effective and cheap when used to prevent such adhesions in humans. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Adhesion Allium sativum Garlic Peritoneal Prevention Postoperative
1. Introduction Postoperative peritoneal adhesion (PPA), defined as development of fibrous bands between peritoneal surfaces, is a serious and common (90% of patients) complication of abdominopelvic surgery [1,2]. PPA is triggered by disruption of the normal healing process. Infection, ischemia, hemorrhage, trauma, and foreign body reaction are the principal causes of adhesion formation after surgery [2]. PPA causes various health problems, including bowel obstruction and chronic pelvic pain, and is also a leading cause of female
* Corresponding author. Tel.: +90 5054439900. E-mail address: [email protected] (A. Sahbaz). http://dx.doi.org/10.1016/j.ejogrb.2014.03.018 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
infertility [2–4]. PPA is devastating to millions and a major financial burden on healthcare systems. Worldwide, the total annual cost of hospitalization (including surgery) of PPA patients is about $1.3 billion [5]. Anti-inflammatory agents, antioxidants, anticoagulants, and fibrinolytics have been used in efforts to prevent adhesion [6], but no optimal treatment option has yet been identified. Allium sativum (garlic), of the Liliaceae, has been used as both a nutrient and a folk medicine for centuries. Sulfur-containing compounds, including diallyl trisulfide (DAT) and diallyl sulfide (DAD) give garlic its well-known flavor, and exert physiological effects [7,8]. The antibacterial, anti-inflammatory, antithrombotic, fibrinolytic, antioxidant, and wound-healing properties of garlic have been well-documented in previous studies [7–9]. We thus hypothesized that garlic oil might prevent PPA.
A. Sahbaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 177 (2014) 44–47
45
2. Materials and methods
2.1. Adhesion grading
This study was approved by the Ethics Committee for Animal Experiments of Zonguldak Bulent Ecevit University and was performed in the Experimental Animal Production and Research Laboratory of the Medical School of that University, Zonguldak, Turkey. Thirty female Wistar rats (200–250 g, caged in groups of five) were used. Rats were fed ad libitum with standard pelleted food approved by the Turkish Standards Institute and had free access to tap water. The rats were randomly divided into three groups of 10 rats each. Group 1 rats received 5 ml/kg garlic oil (Oleum Allii SativiR; Arifoglu, Firuzkoy Esenyurt Yolu No. 32 Avcilar, I˙stanbul) via injection into the peritoneal cavity using 22-French syringes (Fig. 1A). The dose of garlic oil was determined as suggested by Chen et al. [10]. Rats were anesthesized by intramuscular injection of ketamine (75 mg/kg; Ketalar; Parke-Davis and Co. Inc., Detroit, MI) and 40 mg/kg xylazine (Rompun; Bayer, Berlin, Germany). Each rat was placed on its back on an operating table. The abdominal skin was sterilized with povidone iodine (Betadine; Kurtsan Co., Istanbul, Turkey) and a vertical midline incision (3 cm long) was made. The terminal ileum and cecum were mobilized and draped over the left index finger of the surgeon. The anterior surface of each cecum was scraped with dry gauze to induce serosal petechiae (Fig. 1B) and arteries supplying the scraped segment were clamped for 1 min to induce transient ischemia (the ‘‘scraping model’’) [11]. The cecum was replaced and the incision closed with continuous 3-0 polypropylene sutures (Prolene; Bicakcilar Co., Istanbul, Turkey). Group 2 (control) rats received no treatment. Group 3 (test) rats were treated with 5 ml/kg garlic oil, delivered into the peritoneal cavity immediately after scraping. All rats were next individually caged, weighed regularly, and observed closely for development of surgical complications. Ten days later, all animals were sacrificed via intraperitoneal injection of a high dose of sodium pentothal. Reverse-U incisions were made to allow of maximal exploration.
The modified Evans adhesion scoring system, which is simple and logical, was used [12]. In this system, grade 0 = no adhesions; grade 1 = smooth adhesions that split either spontaneously or upon application of weak traction; grade 2 = firm adhesions splitting when traction is applied; and grade 3 = dense adhesions requiring dissection with a blade [12]. The same researcher, blinded to Group, determined all adhesion scores. The strongest adhesion identified in a rat defined the adhesion grade of that rat (Fig. 2).
[(Fig._1)TD$IG]
Fig. 1. (A) Intraperitoneal administration of garlic oil. (B) Serosal petechiae.
2.2. Histopathologic evaluation The cecal area and associated adhesive lesions were resected and preserved in formalin solution. After routine histological examination process, 5 mm thick sections were prepared and stained with hematoxylin and eosin. Under a light microscope, the histological sections were examined and scored for the presence of inflammatory cell infiltrate (0: absent or normal in number, 1: slight increase, 2: moderate infiltration, 3: massive infiltration), fibrosis (0: none, 1: slight, 2: moderate, 3: dense) and vascularization (0: none, 1: one to two vessels, 2: three to nine vessels, 3: ten or more vessels) (Fig. 3). Statistical analysis was performed using the MedCalc software (version 11.5 for Windows). The nonparametric Mann–Whitney Utest was used to explore the significance of among-Group differences in the grading of adhesions. A p value < 0.05 was considered ‘‘significant’’ and p < 0.001 ‘‘highly significant’’.
3. Results All animals tolerated the operations well. No signs of infection were observed in the incision site throughout the study period. No adhesions were evident upon laparotomy of Group 1 animals (no operation, but intraperitoneal admistration of garlic oil to evaluate any adhesion formation related to garlic oil use). In Group 2 three rats had an adhesion grade 2 and seven rats had an adhesion of grade 3, whereas in Group 3 no adhesions were found in four rats,
[(Fig._2)TD$IG]
Fig. 2. Macroscopic scores of adhesions. (A) Grade 0. (B) Grade 1. (C) Grade 2. (D) Grade 3.
[(Fig._3)TD$IG]
A. Sahbaz et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 177 (2014) 44–47
46
neovascularization scores between Group 2 and 3 (p = 0.001, p = 0.001, p = 0.011 respectively). Inflammation, fibrosis and vascularization scores in Group 3 were found significantly lower than Group 2. 4. Comments
Fig. 3. (a) Grade 3 inflammation with giant cell formation (H&E, 40). (b) Capillary vessel proliferation (grade 2 neovascularization) (H&E, 100). (c) Moderate fibrosis (grade 2) represented by young fibroblasts (H&E, 200).
five rats had an adhesion grade of 1. Only one rat had a grade 2 adhesion. Macroscopic adhesions and mean adhesions scores of the groups are summarized in Table 1. The adhesion scores of Group 3 were significantly lower compared to Group 2 (p < 0.001). Histopathologic evaluation scores of the groups with respect to inflammation, fibrosis and neovascularization are shown in Table 1. Histopathologic evaluation of the specimens also revealed a statistically significant differences in inflammation, fibrosis, and Table 1 Mean adhesion, inflammation, fibrosis, neovascularization scores. Group 2 n = 10
Group 3 n = 10
p value
2.70 0.48
0.70 0.67
