Journal of Investigative Surgery, Early Online, 1–8, 2014 C 2014 Informa Healthcare USA, Inc. Copyright ISSN: 0894-1939 print / 1521-0553 online DOI: 10.3109/08941939.2014.908988
ORIGINAL ARTICLE
Intraperitoneal Nigella sativa for Prevention of Postoperative Intra-Abdominal Adhesions in Rats
J Invest Surg Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 06/21/14 For personal use only.
Ahmet Karatas,1 Tulay Ozlu,1 Gulzade Ozyalvacli,2 Mehmet Tosun,3 Ayhan Cetinkaya,4 Melahat Emine Donmez,1 Arzu Turker,5 Husna Bayrakdar2 1
Department of Obstetrics and Gynecology, Abant Izzet Baysal University Medical School, Bolu, Turkey, 2 Department of Medical Pathology, Abant Izzet Baysal University Medical School, Bolu, Turkey, 3 Department of Medical Biochemistry, Abant Izzet Baysal University Medical School, Bolu, Turkey, 4 Experimental Animals Application and Research Center, Abant Izzet Baysal University, Bolu, Turkey, 5 Department of Biology, Abant Izzet Baysal University Faculty of Science, Bolu, Turkey
ABSTRACT Objective: To evaluate intraperitoneal administration of Nigella sativa (NS) to prevent postoperative intraperitoneal adhesion (PPA) after surgical manipulation of rat uterine horn. Materials and Methods: Two forms of NS were used in the study (Volatile oil (NSVO) and the ethanolic extract (NSEE)). A total of 50 rats were randomly assigned to the sham group (n = 10), control group (n = 10), NSVO group (n = 10), NSEE group (n = 10), and the Seprafilm group(n = 10). After 14 days, rats were sacrificed. Adhesions were examined macroscopically, and degree of adhesions was scored. A part of horn was excised, and superoxide dismutase (SOD) and glutathione peroxidase activities as well as malondialdehyde levels were evaluated, and histological score was calculated. Results: Total microscopic score of the NSEE group was significantly lower than the control group (p = .001) and was marginally significantly lower than the seprafilm group (p = .005). Collagen formation score was higher in the seprafilm group compared to the sham and NSEE groups (p < 0.001, p = .003, respectively). Apoptotic cells were lower in the NSEE group compared to the control group (p = .003) and also lower in the NSEE and NSVO groups compared to the seprafilm group (p = .001, p < .001, respectively). Only SOD activity was higher in the NSVO and seprafilm groups compared to the control group (p < .001). Conclusion: NSEE form seems to have a possible effect in the prevention of PPAs. This may occur by its effect in decreasing collagen formation and by decreasing apoptosis in the injured tissues. NSVO form seems to induce SOD. Therefore, combined use of NSVO with seprafilm may increase the adhesion preventive effect of seprafilm. Keywords: adhesion; Nigella sativa; peritoneal; postoperative; ethanolic extract; volatile oil
INTRODUCTION
and yet, there is no universally accepted application to prevent PPAs. Sodium hyaluronate carboxymethylcellulose (HA/ R ), CMC)-based bioresorbable membrane (Seprafilm one of the adhesion barriers approved by FDA, has satisfactory biocompatibility, adherence to tissue, low side effect profile, optimal reabsorbing time at surgical surface but high cost and difficult handling. Some studies show that seprafilm was inadequate in eliminating the adhesions [4, 5], however, randomized controlled studies report its efficacy [6, 7]. Its disadvantages include being very vulnerable to
Pelvic adhesions after gynecologic surgeries may lead to infertility, ectopic pregnancy, deep dyspareunia, chronic pelvic pain, bowel obstructions, and difficult subsequent surgeries. Adhesions occur with an incidence of 55%–94% in patients having open surgery [1]. About one-third of patients have an average of two readmissions during the following 10 years after an abdominal or pelvic surgery because of adhesionrelated problems [2, 3]. Postoperative peritoneal adhesions (PPAs) is an unresolved problem for surgeons
Received 1 January 2014; accepted 24 March 2014. Address correspondence to Ahmet Karatas, Abant Izzet Baysal University Medical School, Department of Obstetrics and Gynecology, 14280, Golkoy, Bolu, Turkey. E-mail: [email protected]
1
2 A. Karatas et al.
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tears and being difficult to handle, and it easily adheres to gloves and wounds besides the target organ [8]. Nigella sativa (NS) has various biological activities such as immunomodulatory, anti-inflammatory, antioxidant, analgesic, antiviral, and antineoplastic effects [9–15]. It contains more than 30 of a fixed oil and 0.40–0.45w/w of a volatile oil (18.4%–24% thymoquinone and 46% monoterpenes) which are responsible for its mentioned effects. So, we can suggest that it may also be effective in the prevention of PPAs. In vivo efficacy of NS in the prevention of PAPs was first demonstrated in a very recent study [16]. Here, we also aimed to evaluate the efficacy of intraperitoneal administration of NS to prevent PPAs.
MATERIALS AND METHODS This study was carried out in the Animal Research Laboratory of Abant Izzet Baysal University. Approval from the Animal Ethics Committee of Abant Izzet Baysal University was obtained before the study. The study was carried out according to the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. A total of 50 female Wistar Albino rats (body weight, 200–250 g) were assigned into five groups, each containing 10 rats. The rats were housed in individual cages on a 12:12 light/dark schedule under controlled temperature (21–23◦ C) and a relative humidity of 60%–65%, and they had free access to standard rat diet and water. Before operation, the day of estrus cycle for the rats was noted. All rats were operated on the same cycle day. They were anesthetized with ketamine (Richter Pharma AG)/xylazine (rompun 2%, Bayer, Germany) (90 mg/kg intraperitoneally (i.p.) and 10 mg/kg (i.p.)). The abdominal area was shaved and then scrubbed with povidone iodine solution (Diagnokim Tıbbi Urunler, Istanbul, Turkey) before surgery. A 3-cm midline vertical abdominal incision was performed, uterine horns were exposed, and a 2-cm segment of the antimesenteric site of the right uterine horn was traumatized by employing bipolar electrocautery in 10 spots of 1–2 mm diameter using 10 W for 1 s for each lesion [17]. The left uterine horn of each rat served as the control group. Nigella sativa forms containing the active ingredients (volatile oil or ethanol extraction forms) can be prepared by different methods. Different forms may have different levels of effects [18, 19]. In the present study, we evaluated the efficacy of both forms to prevent PPAs. NS seeds obtained from the herbalist was used to prepare the ethanolic extract (EE) and volatile oil (VO) forms.
Preparation of the Ethanolic Extract of NS The extract was prepared from 100 g NS seed and 350 ml of ethanol at 75◦ C by using the soxhlet extractor.
At the end of 24 hr, the solution was filtered and the liquid component was evaporated by using the rotatory evaporator. The extract remaining at the balloon was dissolved in 20–30 ml of distilled water, the liquid component (water) was evaporated by using the lyophilizer, and the extract was obtained as a powder form.
Preparation of the Volatile Oil of NS Hundred grams of NS seed was powderized, then loaded to the balloon in the Clevenger system, and adequate water was added above it. The Clevenger system was run at 100◦ C for 3–4 hr. Volatile oil of the NS seeds was obtained finally. Both the EE and the VO were dissolved in sterile physiologic saline (0.9%) which were applied to the rats at a dose of 0.2 ml/kg [20]. Group 1 (sham group) underwent laparotomy only, without intraperitoneal administration of any substance. In group 2 (control group), 1 ml of 0.9% sodium chloride was instilled onto the right uterine horn, just before the closure of the laparatomy incision. In group 3 (NS volatile oil (NSVO) group), 1 ml NSVO was instilled onto the right uterine horn. In group 4 (NSethanol extraction (NSEE) group), 1 ml NSEE form, was instilled on to the right uterine horn. In group R 5 (Seprafilm group) a 20×30-mm sheet of Seprafilm material was applied directly to the right uterine horn before closure. The abdominal incision was then closed in two layers with continuous 3/0 delayed absorbable suture. We did not administer any additional analgesic and antibiotics before or after the procedure. All rats were allowed to resume their diets until the 14th postoperative day. The peritoneal cavities were reentered via a reversed U-shaped incision on the anterior abdominal wall above the prior incision. The abdominal wall was retracted caudally to provide maximal exposure. Adhesions were examined macroscopically and degree of adhesions was scored by one of the investigators who was blinded to the groups according to the Blauer and Collins scale [21] as follows: 0, no adhesion; 1, thin adhesive bands, easily removable; 2, thick adhesive bands limited to one area; 3, extensive and thick adhesive bands; and 4, extensive and thick adhesive bands and adhesions between viscera and/or abdominal wall (Figure 1). A part of the right uterine horn and the adhesion tissue (if present) was excised and stored at −80◦ C for biochemical analyses and another part was preserved in 10% formalin for histopathological examination. All rats were sacrificed by exsanguinations. Biochemical Analysis In the tissues obtained from the right uterine horn and adhesions, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities as well as malondialdehyde (MDA) levels were evaluated. Tissue Journal of Investigative Surgery
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Nigella sativa to Prevent Pelvic Adhesions
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FIGURE 1. Macroscopic adhesion score. (A) Grade 1; thin adhesive bands, easily removable, (B) Grade 2; thick adhesive bands limited to one area, (C) Grade 3; extensive and thick adhesive bands, (D) Grade 4; extensive and thick adhesive bands and adhesions between viscera and/or abdominal wall.
Cu-Zn SOD activity and tissue GPx activity were measured by specific enzyme-linked immunosorbant assays (EIAab [Wuhan EIAab Science Co., Ltd; Wuhan, P.R.China]) Tissue MDA levels were determined by using specific enzyme-linked immunosorbant assay (Cusabio [Hubei Province, P.R.China]).
Histopathological Examination Tissues containing adhesions were excised en bloc and the samples were fixed in a 10% formaldehyde solution. Samples were routinely processed by dehydration and paraffin embedding, and 5-μm cross-sections were prepared using a microtome. Samples were examined under light microscope after hemotoxylin-eosin staining, and scored according to the inflammation, C
2014 Informa Healthcare USA, Inc.
fibroblastic activities, foreign body reaction, collagen formation, and vascular proliferation (Figure 2). As indicated in Table 1, the scoring was classified into five groups. Thus, the total histological score of each rat was calculated according to a semiquantitative method as mentioned [22]. Tunel staining was evaluated blindly by the same pathologist (GO) to determine the apoptosis. This was performed by counting the number of stained epithelial cells among 100 epithelial cells at randomly selected three sites and by taking the average of these (Figure 3).
Statistical Analysis The data were analyzed by using SPSS for Windows version 15.0. Normality of distribution of the continuous variables was evaluated by using the Shapiro Wilk
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4 A. Karatas et al.
FIGURE 2. Photographs of hematoxylin-eosin stainings for microscopic adhesion score according to the inflammation, fibroblastic activities, foreign body reaction, collagen formation, and vascular proliferation. (A) Grade 0, (B) Grade 1, (C) Grade 2, (D) Grade 3. (A, B, C, D; 10× original magnification) (22).
test. Descriptive statistics of the continuous and ordinal data were expressed as median (minimum–maximum). Significance of the between-group differences of the median values was evaluated by the Kruskal Wallis test. P < 0,05 was considered to be statistically significant. When a statistically significant result was obtained at the Kruskal Wallis test, the causes of the difference were determined by using the Mann–Whitney U test with Bonferroni correction and P < 0,005 was considered to be statistically significant.
TABLE 1.
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4
RESULTS A total of 50 rats were randomly assigned to the sham group (n = 10), control group (n = 10), NSVO group (n = 10), NSEE group (n = 10), and the Seprafilm group (n = 10). Two rats (one in the NSVO group and one in the Seprafilm group) died before re-laparotomy. These rats were replaced with new rats; the steps of the study for the corresponding groups were performed for the new rats to be included in the study.
Histological scoring system [16] Inflammation
Fibroblastic activity
Foreign body reaction
Collagen formation
Vascular proliferation
No inflammation (25%) mixed inflammation (50%) mixed inflammation (75%) mixed inflammation Massive inflammation
No Mild Moderate Marked Massive
No Rare Few Several Numerous and abnormally shaped
No Mild Moderate Marked Massive
No Mild Moderate Marked Massive
Journal of Investigative Surgery
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Nigella sativa to Prevent Pelvic Adhesions
5
FIGURE 3. Photographs of TUNEL stainings. (A) Negative staining, (B) Positive staining. (A, B; 20× original magnification).
The intra-abdominal macroscopic adhesions and histopathological fibrosis scores in all groups are summarized in Table 2. The macroscopic score of the sham group was higher than the other four groups and it was similar between the study and the control groups. Total microscopic score of the NSEE group was significantly lower than the control group (p = .001) and was marginally significantly lower than the seprafilm group (p = .005). Histological scoring was also performed according to inflammation, fibroblastic activity, foreign body reaction, collagen formation, and vascular proliferation. Inflammation, fibroblastic activity, foreign body reaction, and vascular proliferation were found to be similar between study and control groups (Table 2). Foreign body reaction score was lower in the sham group than the other four groups. The most prominent difference from the sham group of the foreign body reaction score was observed in the NSVO group (p = .007) but this difference was not found to be significant after the Bonferroni correction. Collagen formation score was higher in the seprafilm group compared to the sham and NSEE groups (p < .001, p = .003, respectively). Apoptotic cells TABLE 2.
were lower in the NSEE group compared to the control group (p = .003) and they were also lower in the NSEE and NSVO groups compared to the seprafilm group (p = .001 and p < .001, respectively). Between-group comparisons of SOD and GSH-Px enzyme activities and MDA levels are shown in Table 3. SOD enzyme activity was higher in the NSVO group compared to the control group, and it was higher in the seprafilm group compared to the sham and control groups (p < 0.001 for all). GSH-Px enzyme activity and MDA levels were similar between the groups (p = .438 and p = .024, respectively).
DISCUSSION Adhesions are the result of tissue injuries that may occur by cutting, mechanical or thermal injury, infection, radiation, ischemia, venous obstruction, drying, abrasions, and blood or foreign body reactions. The process of adhesions starts with tissue inflammation; fibrin is deposited within the inflammatory exudate. Physical, chemical, or ischemic trauma decreases the fibrinolytic
Comparison of the adhesion scores and histological features between groups
Macroscopic score Microscopic score Inflammation Fibroblastic activity Foreign body reaction Collagen formation Vascular proliferation Apoptotic cell (%)
Sham (n = 10)
Control (n = 10)
NS-etanol (n = 10)
NS-volatile (n = 10)
Seprafilm (n = 10)
P value∗
0 0 1 (0–2) 1 0 1 (1–2) 2 (1–3) 1.5 (0–15)
2 (0–4) 2 (1–3) 1.5 (1–3) 1 (1–2) 0 (0–2) 1.5 (1–3) 2 (1–2) 8.5 (3–25)
1.5 (0–3) 1 (0–2) a 1 (0–2) 1 (0–2) 0 (0–2) 1 (0–2) 2 (0–2) 1 (0–6)d
2 (0–3) 2 (1–2) 2 (1–2) 1 (1–2) 1.5 (0–3) 1.5 (1 -2) 2 (1–2) 3.5 (2–5)
3 (1–4) 2 (1–3) 2 (1–3) 1 (1–2) 1 (0–3) 2 (2–3)b,c 2 (1–3) 11.5 (5–15)e,f
0.001 < 0.001 0.024 0.815 0.003 0.001 0.444 < 0.001
∗ Kruskal-Wallis Test. The superscripts indicate a statistically significant differences according to Bonferroni corrected Mann Whitney U tets.. Data are presented as median (minimum–maximum). a NS-etanol vs. control (p = .001), b Seprafilm vs. sham (p
ORIGINAL ARTICLE
Intraperitoneal Nigella sativa for Prevention of Postoperative Intra-Abdominal Adhesions in Rats
J Invest Surg Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 06/21/14 For personal use only.
Ahmet Karatas,1 Tulay Ozlu,1 Gulzade Ozyalvacli,2 Mehmet Tosun,3 Ayhan Cetinkaya,4 Melahat Emine Donmez,1 Arzu Turker,5 Husna Bayrakdar2 1
Department of Obstetrics and Gynecology, Abant Izzet Baysal University Medical School, Bolu, Turkey, 2 Department of Medical Pathology, Abant Izzet Baysal University Medical School, Bolu, Turkey, 3 Department of Medical Biochemistry, Abant Izzet Baysal University Medical School, Bolu, Turkey, 4 Experimental Animals Application and Research Center, Abant Izzet Baysal University, Bolu, Turkey, 5 Department of Biology, Abant Izzet Baysal University Faculty of Science, Bolu, Turkey
ABSTRACT Objective: To evaluate intraperitoneal administration of Nigella sativa (NS) to prevent postoperative intraperitoneal adhesion (PPA) after surgical manipulation of rat uterine horn. Materials and Methods: Two forms of NS were used in the study (Volatile oil (NSVO) and the ethanolic extract (NSEE)). A total of 50 rats were randomly assigned to the sham group (n = 10), control group (n = 10), NSVO group (n = 10), NSEE group (n = 10), and the Seprafilm group(n = 10). After 14 days, rats were sacrificed. Adhesions were examined macroscopically, and degree of adhesions was scored. A part of horn was excised, and superoxide dismutase (SOD) and glutathione peroxidase activities as well as malondialdehyde levels were evaluated, and histological score was calculated. Results: Total microscopic score of the NSEE group was significantly lower than the control group (p = .001) and was marginally significantly lower than the seprafilm group (p = .005). Collagen formation score was higher in the seprafilm group compared to the sham and NSEE groups (p < 0.001, p = .003, respectively). Apoptotic cells were lower in the NSEE group compared to the control group (p = .003) and also lower in the NSEE and NSVO groups compared to the seprafilm group (p = .001, p < .001, respectively). Only SOD activity was higher in the NSVO and seprafilm groups compared to the control group (p < .001). Conclusion: NSEE form seems to have a possible effect in the prevention of PPAs. This may occur by its effect in decreasing collagen formation and by decreasing apoptosis in the injured tissues. NSVO form seems to induce SOD. Therefore, combined use of NSVO with seprafilm may increase the adhesion preventive effect of seprafilm. Keywords: adhesion; Nigella sativa; peritoneal; postoperative; ethanolic extract; volatile oil
INTRODUCTION
and yet, there is no universally accepted application to prevent PPAs. Sodium hyaluronate carboxymethylcellulose (HA/ R ), CMC)-based bioresorbable membrane (Seprafilm one of the adhesion barriers approved by FDA, has satisfactory biocompatibility, adherence to tissue, low side effect profile, optimal reabsorbing time at surgical surface but high cost and difficult handling. Some studies show that seprafilm was inadequate in eliminating the adhesions [4, 5], however, randomized controlled studies report its efficacy [6, 7]. Its disadvantages include being very vulnerable to
Pelvic adhesions after gynecologic surgeries may lead to infertility, ectopic pregnancy, deep dyspareunia, chronic pelvic pain, bowel obstructions, and difficult subsequent surgeries. Adhesions occur with an incidence of 55%–94% in patients having open surgery [1]. About one-third of patients have an average of two readmissions during the following 10 years after an abdominal or pelvic surgery because of adhesionrelated problems [2, 3]. Postoperative peritoneal adhesions (PPAs) is an unresolved problem for surgeons
Received 1 January 2014; accepted 24 March 2014. Address correspondence to Ahmet Karatas, Abant Izzet Baysal University Medical School, Department of Obstetrics and Gynecology, 14280, Golkoy, Bolu, Turkey. E-mail: [email protected]
1
2 A. Karatas et al.
J Invest Surg Downloaded from informahealthcare.com by Technische Universiteit Eindhoven on 06/21/14 For personal use only.
tears and being difficult to handle, and it easily adheres to gloves and wounds besides the target organ [8]. Nigella sativa (NS) has various biological activities such as immunomodulatory, anti-inflammatory, antioxidant, analgesic, antiviral, and antineoplastic effects [9–15]. It contains more than 30 of a fixed oil and 0.40–0.45w/w of a volatile oil (18.4%–24% thymoquinone and 46% monoterpenes) which are responsible for its mentioned effects. So, we can suggest that it may also be effective in the prevention of PPAs. In vivo efficacy of NS in the prevention of PAPs was first demonstrated in a very recent study [16]. Here, we also aimed to evaluate the efficacy of intraperitoneal administration of NS to prevent PPAs.
MATERIALS AND METHODS This study was carried out in the Animal Research Laboratory of Abant Izzet Baysal University. Approval from the Animal Ethics Committee of Abant Izzet Baysal University was obtained before the study. The study was carried out according to the Declaration of Helsinki and the Guiding Principles in the Care and Use of Animals. A total of 50 female Wistar Albino rats (body weight, 200–250 g) were assigned into five groups, each containing 10 rats. The rats were housed in individual cages on a 12:12 light/dark schedule under controlled temperature (21–23◦ C) and a relative humidity of 60%–65%, and they had free access to standard rat diet and water. Before operation, the day of estrus cycle for the rats was noted. All rats were operated on the same cycle day. They were anesthetized with ketamine (Richter Pharma AG)/xylazine (rompun 2%, Bayer, Germany) (90 mg/kg intraperitoneally (i.p.) and 10 mg/kg (i.p.)). The abdominal area was shaved and then scrubbed with povidone iodine solution (Diagnokim Tıbbi Urunler, Istanbul, Turkey) before surgery. A 3-cm midline vertical abdominal incision was performed, uterine horns were exposed, and a 2-cm segment of the antimesenteric site of the right uterine horn was traumatized by employing bipolar electrocautery in 10 spots of 1–2 mm diameter using 10 W for 1 s for each lesion [17]. The left uterine horn of each rat served as the control group. Nigella sativa forms containing the active ingredients (volatile oil or ethanol extraction forms) can be prepared by different methods. Different forms may have different levels of effects [18, 19]. In the present study, we evaluated the efficacy of both forms to prevent PPAs. NS seeds obtained from the herbalist was used to prepare the ethanolic extract (EE) and volatile oil (VO) forms.
Preparation of the Ethanolic Extract of NS The extract was prepared from 100 g NS seed and 350 ml of ethanol at 75◦ C by using the soxhlet extractor.
At the end of 24 hr, the solution was filtered and the liquid component was evaporated by using the rotatory evaporator. The extract remaining at the balloon was dissolved in 20–30 ml of distilled water, the liquid component (water) was evaporated by using the lyophilizer, and the extract was obtained as a powder form.
Preparation of the Volatile Oil of NS Hundred grams of NS seed was powderized, then loaded to the balloon in the Clevenger system, and adequate water was added above it. The Clevenger system was run at 100◦ C for 3–4 hr. Volatile oil of the NS seeds was obtained finally. Both the EE and the VO were dissolved in sterile physiologic saline (0.9%) which were applied to the rats at a dose of 0.2 ml/kg [20]. Group 1 (sham group) underwent laparotomy only, without intraperitoneal administration of any substance. In group 2 (control group), 1 ml of 0.9% sodium chloride was instilled onto the right uterine horn, just before the closure of the laparatomy incision. In group 3 (NS volatile oil (NSVO) group), 1 ml NSVO was instilled onto the right uterine horn. In group 4 (NSethanol extraction (NSEE) group), 1 ml NSEE form, was instilled on to the right uterine horn. In group R 5 (Seprafilm group) a 20×30-mm sheet of Seprafilm material was applied directly to the right uterine horn before closure. The abdominal incision was then closed in two layers with continuous 3/0 delayed absorbable suture. We did not administer any additional analgesic and antibiotics before or after the procedure. All rats were allowed to resume their diets until the 14th postoperative day. The peritoneal cavities were reentered via a reversed U-shaped incision on the anterior abdominal wall above the prior incision. The abdominal wall was retracted caudally to provide maximal exposure. Adhesions were examined macroscopically and degree of adhesions was scored by one of the investigators who was blinded to the groups according to the Blauer and Collins scale [21] as follows: 0, no adhesion; 1, thin adhesive bands, easily removable; 2, thick adhesive bands limited to one area; 3, extensive and thick adhesive bands; and 4, extensive and thick adhesive bands and adhesions between viscera and/or abdominal wall (Figure 1). A part of the right uterine horn and the adhesion tissue (if present) was excised and stored at −80◦ C for biochemical analyses and another part was preserved in 10% formalin for histopathological examination. All rats were sacrificed by exsanguinations. Biochemical Analysis In the tissues obtained from the right uterine horn and adhesions, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities as well as malondialdehyde (MDA) levels were evaluated. Tissue Journal of Investigative Surgery
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Nigella sativa to Prevent Pelvic Adhesions
3
FIGURE 1. Macroscopic adhesion score. (A) Grade 1; thin adhesive bands, easily removable, (B) Grade 2; thick adhesive bands limited to one area, (C) Grade 3; extensive and thick adhesive bands, (D) Grade 4; extensive and thick adhesive bands and adhesions between viscera and/or abdominal wall.
Cu-Zn SOD activity and tissue GPx activity were measured by specific enzyme-linked immunosorbant assays (EIAab [Wuhan EIAab Science Co., Ltd; Wuhan, P.R.China]) Tissue MDA levels were determined by using specific enzyme-linked immunosorbant assay (Cusabio [Hubei Province, P.R.China]).
Histopathological Examination Tissues containing adhesions were excised en bloc and the samples were fixed in a 10% formaldehyde solution. Samples were routinely processed by dehydration and paraffin embedding, and 5-μm cross-sections were prepared using a microtome. Samples were examined under light microscope after hemotoxylin-eosin staining, and scored according to the inflammation, C
2014 Informa Healthcare USA, Inc.
fibroblastic activities, foreign body reaction, collagen formation, and vascular proliferation (Figure 2). As indicated in Table 1, the scoring was classified into five groups. Thus, the total histological score of each rat was calculated according to a semiquantitative method as mentioned [22]. Tunel staining was evaluated blindly by the same pathologist (GO) to determine the apoptosis. This was performed by counting the number of stained epithelial cells among 100 epithelial cells at randomly selected three sites and by taking the average of these (Figure 3).
Statistical Analysis The data were analyzed by using SPSS for Windows version 15.0. Normality of distribution of the continuous variables was evaluated by using the Shapiro Wilk
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4 A. Karatas et al.
FIGURE 2. Photographs of hematoxylin-eosin stainings for microscopic adhesion score according to the inflammation, fibroblastic activities, foreign body reaction, collagen formation, and vascular proliferation. (A) Grade 0, (B) Grade 1, (C) Grade 2, (D) Grade 3. (A, B, C, D; 10× original magnification) (22).
test. Descriptive statistics of the continuous and ordinal data were expressed as median (minimum–maximum). Significance of the between-group differences of the median values was evaluated by the Kruskal Wallis test. P < 0,05 was considered to be statistically significant. When a statistically significant result was obtained at the Kruskal Wallis test, the causes of the difference were determined by using the Mann–Whitney U test with Bonferroni correction and P < 0,005 was considered to be statistically significant.
TABLE 1.
Grade 0 Grade 1 Grade 2 Grade 3 Grade 4
RESULTS A total of 50 rats were randomly assigned to the sham group (n = 10), control group (n = 10), NSVO group (n = 10), NSEE group (n = 10), and the Seprafilm group (n = 10). Two rats (one in the NSVO group and one in the Seprafilm group) died before re-laparotomy. These rats were replaced with new rats; the steps of the study for the corresponding groups were performed for the new rats to be included in the study.
Histological scoring system [16] Inflammation
Fibroblastic activity
Foreign body reaction
Collagen formation
Vascular proliferation
No inflammation (25%) mixed inflammation (50%) mixed inflammation (75%) mixed inflammation Massive inflammation
No Mild Moderate Marked Massive
No Rare Few Several Numerous and abnormally shaped
No Mild Moderate Marked Massive
No Mild Moderate Marked Massive
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Nigella sativa to Prevent Pelvic Adhesions
5
FIGURE 3. Photographs of TUNEL stainings. (A) Negative staining, (B) Positive staining. (A, B; 20× original magnification).
The intra-abdominal macroscopic adhesions and histopathological fibrosis scores in all groups are summarized in Table 2. The macroscopic score of the sham group was higher than the other four groups and it was similar between the study and the control groups. Total microscopic score of the NSEE group was significantly lower than the control group (p = .001) and was marginally significantly lower than the seprafilm group (p = .005). Histological scoring was also performed according to inflammation, fibroblastic activity, foreign body reaction, collagen formation, and vascular proliferation. Inflammation, fibroblastic activity, foreign body reaction, and vascular proliferation were found to be similar between study and control groups (Table 2). Foreign body reaction score was lower in the sham group than the other four groups. The most prominent difference from the sham group of the foreign body reaction score was observed in the NSVO group (p = .007) but this difference was not found to be significant after the Bonferroni correction. Collagen formation score was higher in the seprafilm group compared to the sham and NSEE groups (p < .001, p = .003, respectively). Apoptotic cells TABLE 2.
were lower in the NSEE group compared to the control group (p = .003) and they were also lower in the NSEE and NSVO groups compared to the seprafilm group (p = .001 and p < .001, respectively). Between-group comparisons of SOD and GSH-Px enzyme activities and MDA levels are shown in Table 3. SOD enzyme activity was higher in the NSVO group compared to the control group, and it was higher in the seprafilm group compared to the sham and control groups (p < 0.001 for all). GSH-Px enzyme activity and MDA levels were similar between the groups (p = .438 and p = .024, respectively).
DISCUSSION Adhesions are the result of tissue injuries that may occur by cutting, mechanical or thermal injury, infection, radiation, ischemia, venous obstruction, drying, abrasions, and blood or foreign body reactions. The process of adhesions starts with tissue inflammation; fibrin is deposited within the inflammatory exudate. Physical, chemical, or ischemic trauma decreases the fibrinolytic
Comparison of the adhesion scores and histological features between groups
Macroscopic score Microscopic score Inflammation Fibroblastic activity Foreign body reaction Collagen formation Vascular proliferation Apoptotic cell (%)
Sham (n = 10)
Control (n = 10)
NS-etanol (n = 10)
NS-volatile (n = 10)
Seprafilm (n = 10)
P value∗
0 0 1 (0–2) 1 0 1 (1–2) 2 (1–3) 1.5 (0–15)
2 (0–4) 2 (1–3) 1.5 (1–3) 1 (1–2) 0 (0–2) 1.5 (1–3) 2 (1–2) 8.5 (3–25)
1.5 (0–3) 1 (0–2) a 1 (0–2) 1 (0–2) 0 (0–2) 1 (0–2) 2 (0–2) 1 (0–6)d
2 (0–3) 2 (1–2) 2 (1–2) 1 (1–2) 1.5 (0–3) 1.5 (1 -2) 2 (1–2) 3.5 (2–5)
3 (1–4) 2 (1–3) 2 (1–3) 1 (1–2) 1 (0–3) 2 (2–3)b,c 2 (1–3) 11.5 (5–15)e,f
0.001 < 0.001 0.024 0.815 0.003 0.001 0.444 < 0.001
∗ Kruskal-Wallis Test. The superscripts indicate a statistically significant differences according to Bonferroni corrected Mann Whitney U tets.. Data are presented as median (minimum–maximum). a NS-etanol vs. control (p = .001), b Seprafilm vs. sham (p
