Histopathological assessment of nano n-acetyl cysteine effect on postoperative adhesion in rats

S. Shahzamani¹, A. Jahandideh¹, G. Abedi¹, A. Akbarzadeh², S. Hesaraki³ and P. Parsaei⁴

1Department of Clinical Science, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, 2Universal Scientific Education and Research Network (USERN), 3Department of Pathobiology, Faculty of Specialized Veterinary Sciences, Science and Research Branch, Islamic Azad University, Tabriz, 4Department of Food Hygiene, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

shadmanshahzamani@yahoo.com, 0000-0002-1937-0592

dr.jahandideh@gmail.com, 0000-0002-4212-6416

abedi.gholamreza@yahoo.com, 0000-0001-7743-4227

dr.akbarzadeh2010@gmail.com, 0000-0001-9941-0357

hesarakisaeed@yahoo.com, 0000-0001-7756-5315

pouyaparsaei@yahoo.com, 0000-0001-8105-3697

2020-03-28

2020-06-19

Abstract

Abdominal adhesion is one of the main and important post-surgical problems and, according to available literature, the probability of their occurrence after surgery is higher than 50%. The present study intended to estimate the effect of Nano n-acetylcysteine on macroscopic and microscopic features post-surgical adhesions in an animal model. Twenty-five adult male Wistar rats, 200-220 gr, were randomly divided into five equal groups and underwent intramuscular anesthesia by injection of ketamine 10% at 50 mg/kg and injection of xylazine 2% at 5 mg/ kg. The treatments included three Nano-NAC groups at different dosages 50, 75 and 150 mg/kg, an NAC group, and a control group. On days 14 and 28 post-surgery, the abdomens of all rats were re-opened for macroscopic and microscopic evaluation of adhesions. Based on the results of macroscopic evaluation, the group receiving Nano-NAC 50 mg/kg had the lowest and the control group the highest mean adhesion scores on days14 and 28 post-operations. Histopathological evaluation also revealed that the group treated with Nano-NAC 50 mg/kg had the lowest level of inflammation and fibrosis score among the five groups on days 14 and 28 post-operation. Considering the results obtained in our experimental model and the increasing use of nanotechnology in the manufacture of biomedical drugs, it seems that use of Nano-NAC at low doses 50 and 75mg/kg after laparotomy can significantly reduce adhesions.

Keywords: Nano N-acetylcysteine, Intra-abdominal Adhesions, Histopathology, Rat

Introduction

Abdominal adhesion is one of the common clinical and surgical problems in humans and animals. It is estimated that postoperative adhesions happen in at least 90% of all patients undergoing abdominal surgery (1). Intra-abdominal adhesions result in critical conditions including severe abdominal pain, bowel obstructions and infertility and in serious conditions such as peritonitis they can lead to septicemia and even death (2). Available documents show that about one third of the patients that have undergone abdominal surgery require secondary surgery to remove the adhesions and More than 25% of infertility is related to pelvic or abdominal adhesions. Also, obstruction of the intestines can be the cause of 60 to 70% of all deaths and morbidity (3). Therefore, one of the problems always faced by veterinary and human surgeons is to develop a method that prevents occurrence of abdominal adhesions. Although the main mechanisms responsible for the development of adhesions still remain unclear, they may be caused by factors such as inflammation, bleeding, ischemia, surgical injuries, foreign bodies (such as talc powder and suture material), chemical and thermal injuries, different anastomose techniques, suture methods and genetic predisposition (3). Such traumas can injure peritoneal surfaces triggering histamine release that results in vasodilatation and exudate secretion. The exudate secreted inside the abdominal cavity is the main bedding site for creating adhesions in this area. A literature review shows that numerous local or systemic effect anti-histamine and anti-inflammatory drugs including dexamethasone, aspirin, phospholipase inhibitors, dextran, corticosteroids, and heparin have been assessed for preventing postoperative adhesions. However, none of them has been satisfactory (3,4). N-acetylcysteine (NAC) is one of the most commonly used antioxidants in human and veterinary medicine. It can be used as a specific antidote for acetaminophen poisoning. It can also prevent ischemia/reperfusion injury and modify tumorigenesis (5). NAC contains a free thiol group. It was originally administered as a mucolytic agent by using a nebulizer. NAC is a precursor of L-cysteine that results in glutathione elevation biosynthesis. Therefore, it is primarily used as a classical antioxidant (5). Evaluations to determine the efficacy of NAC in preventing intra-abdominal adhesions showed that its intraperitoneal or oral administration was able to significantly reduce intra-abdominal adhesions. However, the exact dose of NAC to use is still unknown, and new technologies such as nanotechnology are not employed in its administration. Therefore, the present study evaluated the effect of Nano- NAC on reducing adhesions (6). Nanoparticles (NPs) are very tiny particles with diameter

Consequently, the present research utilized nanotechnology to convert NAC to nanoparticles for the first time and gross and histopathological investigations after adding Nano-NAC for reducing post-surgical adhesions in rats.

Materials and methods

This research was conducted at the Faculty of Specialized Veterinary Sciences, Science and Research Branch, Tehran, Iran, with the permission of the Ethics Committee of Islamic Azad University Science and Research Branch. All animals' welfare regulations and policies were fully complied with on the basis of the Ethics Committee Regulations and Letter No. 139605 of Islamic Azad University of Tehran.

Surgical procedure

Twenty-five male Wistar albino rats (mean age: 4 months; mean weight: 200-250g) were used. The experimental animals were randomly divided into five equal groups: the first three groups were treated with different dosage of Nano-NAC 50 mg, Nano-NAC 75 mg, and Nano-NAC 150mg, the NAC and the control groups. The rats were left one night without food and then anesthetized by injection of ketamine 10% at 50 mg/kg and injection of xylazine 2% at 5 mg/ kg intramuscularly (8). After a ventral abdominal region was prepared aseptically a surgery technique was used at the surgical site in the midline part of the abdominal region using 2.5% povidone-iodine. A single abdominal incision 3cm in length was made on the midline. After entering the abdomen through the incision, to induce the standard adhesion in inner abdominal wall or in the serosal surface of peritoneal cavity three 2cm incisions were made on the right abdominal wall longitudinally using a surgical blade (No. 4). To ensure the inducing of abdominal adhesion several 2×2cm pieces of abdominal wall were harvested from the peritoneal cavity by using surgical scissors (8). The abdomen was then closed after the surgical procedure using monofilament absorbable 3.0 (USP) vicryl sutures (Supa, Iran) at 1cm intervals, the fascia and midline muscles were sutured with absorbable monofilament 2.0 (USP) vicryl sutures (Supa, Iran) and, finally, the skin was closed using monofilament non-absorbable 3.0 (USP) silk sutures (Supa, Iran). The anesthetized rats were placed under a heat lamp. Their body temperature was monitored regularly and maintained at 36-38°C with the aid of a thermal lamp.

Treatments

Twenty-five male rats were randomized into 5 groups of 5 and underwent primary surgical procedure. The induced peritoneal adhesions were treated using intra-peritoneal administration of Nano NAC at five different concentrations. The study groups were as follows: (A) the Nano N-acetyl cysteine (Nano-NAC) 50 mg, (B) the Nano-NAC 75 mg, (C) the Nano- NAC 150 mg, (D) the NAC, and (E) the no-treatment (control) group.

Macroscopic evaluation

On days 14 and 28 post-surgery, the abdomen was reopened and the adhesions were examined blindly by the same surgeon and the adhesion rating was determined. To compare the adhesions, the severity of each one was determined separately based on the following scale (Table 1) (9):

Table 1: Lorenz et al (1997) score, macroscopic classification of abdominal adhesions in the rats (9)

Histopathological evaluation

For histopathological examination on days 14 and 28 post-operation, a sample was separated from the adhesive tissue and placed in 10% neutral buffered formalin for 2 days. The samples were prepared for histopathological investigation. Tissue sections (4µm) were routinely stained with Harri's hematoxylin and alcoholic eosin and the stained slides were examined at 100X (10, 8). Adhesions were graded based on the intensity of fibrosis and inflammation according to the following scale (8) separately for each existing adhesion (Table 2).

Table 2:Histopathological criteria for grading adhesions

Data analysis

Data were analyzed and reported only for the rats that completed the trial. Statistical analyses were performed using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA). Severity and fibrosis and inflammation in the abdominal wall surface were reported. All values were expressed as mean+ SEM. Statistical analysis was performed using the ANOVA test, Bartlett test, and Student-Newman-Keuls test (10,11).

Results

Macroscopic assessment results: None of the rats died along the period of treatment. After the operations, all treated rats exhibited healthy. The necrotic lesions appeared on the liver surface of all the members in the Nano-NAC 150 mg and 75 mg groups are higher than those of other groups (Figures 1 and 2). Not only was the drug effective in reducing adhesions at lower doses but there were no necrotic lesions either (Figure3). Our macroscopic assessment of adhesion severity showed that there were statistically significant differences among the five groups. Macroscopic adhesion scores on day 14 post-operation were the lowest in the Nano-NAC 50 mg group (1 in 60% and 0 in 40% of the members) (Figure 4). However, the worst scores were observed in the control group (2 in 60%, 3 in 20%, and 4 in 20% of the members) (Figure5), indicating that there were significant differences between the control and the Nano-NAC 50 mg group (P=0.087) (Tabl. Macroscopic results on day 28 post-operation showed that the lowest adhesion score was that of the Nano-NAC 50 mg group because in none of the members was any adhesion observed, whereas the worst result and the highest adhesion scores were those of the control group in which 40% of the members received the score zero 0, 40% the score 1, and 40% the score 2. The ANOVA test revealed that there was a significant difference between the Nano-NAC 50 mg/kg, NAC and the control on day 28 post-operation (P=0.087). Among the Nano-NAC groups that received different doses of the drug, the best macroscopic results belonged to the Nano-NAC 50 mg and 75 mg groups on days 14 and 28 post-operation with no statically significant differences between them. The only difference between these two groups was that necrotic lesions in the Nano-NAC 75 mg group but not in the Nano-NAC50mg group (Figures2 and 3).

The results of descriptive statistics of histopathological evaluations showed that the inflammation indices on day 14 post-operation in the Nano-NAC 50 mg (mean: 0.400) and Nano-NAC 75 mg (mean: 1.200) were the lowest, whereas in the Nano-NAC 150 mg (Mean: 2.800) and in the control (mean: 2.400) groups they were the highest (Table 3).

There were also statistically significant differences between the Nano-NAC 50mg and Nano-NAC 150 groups (mean diff: 2.400) and between the Nano-NAC 50 and the control groups (mean diff: 2.000) (P < 0:05). In addition, on day 28 post-operation, the lowest scores belonged to the Nano-NAC 50 mg (mean: 0.0) and the Nano-NAC 75 mg (mean: 0.6000) groups and the highest to the control group (mean: 1.800) and to the Nano-NAC 150 mg (mean: 1.800) group. In addition, there were significant differences between the Nano-NAC 50 mg and the control groups (mean diff: 1.800) and between the Nano-NAC 50 mg and the Nano-NAC 150 mg (1.800) groups (Table 4). Figure 6 presents the graph comparing the average scores for all the studied groups.

Figure 1: Macroscopic image of Nano-NAC (150 mg) group on day 14, with multiple necrotic lesions (arrow).

Figure 2: Macroscopic image of Nano-NAC (75 mg) group on day 14, with fewer necrotic lesions.

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Figure 3: Macroscopic image of Nano-NAC (50 mg) group on day 14, with no necrotic lesions.

Figure 4: Macroscopic image of Nano-NAC (50 mg) group on day 14, with no adhesions.

Figure 5: Macroscopic image of control group on day 14, with severe adhesions.

Descriptive statistics of fibrosis index on day 14 post-operation shows that the lowest scores belonged to the Nano-NAC 50 mg (mean: 0.4000) and Nano-NAC 75 mg (mean: 1.600) groups and the highest to the control (Mean: 2.800) and Nano-NAC 150 mg (Mean: 2.200) groups (Table 5). There were significant differences between the Nano-NAC 50 mg and the control groups (mean diff: 2.400) and between the Nano-NAC 50 mg and the Nano-NAC 150 mg (1.800) (Table 5). In addition, on day 28 post-operation, the lowest scores were those of the Nano-NAC 50 mg (mean: 0.0) and the Nano-NAC 75 mg (mean: 0.6000) groups and the highest those of the control group (Mean: 2.000) and the Nano-NAC 150 mg (mean: 1.400) groups. Moreover, there were significant differences between the Nano-NAC 50 mg and the control groups (mean diff: 2.000) and between the Nano-NAC 50 mg and the Nano-NAC 150 mg groups (1.400) (Table 6). Figure 7 presents the graph comparing the average scores for the studied groups.

Table 3: Statistical comparison of inflammation indices between all groups on day 14

Ⅰ: Nano-NAC 50 mg group. Ⅱ: Nano-NAC 75 mg group. Ⅲ: Nano-NAC 150 mg group. Ⅳ: N-Acetylcysteine group. Ⅴ: control group.

Table 4: Statistical comparison of inflammation indices between all groups on day 28

Ⅰ: Nano-NAC 50 mg group. Ⅱ: Nano-NAC 75 mg group. Ⅲ: Nano-NAC 150 mg group. Ⅳ: N-Acetylcysteine group. Ⅴ: control group.

Figure 6: Graph presenting histopathological assessment according to severity of inflammation (mean statistical results) on days 14 and 28.