Influence of Intestinal Strangulation Release on Ischemia-reperfusion Injury in Sprague Dawley Rats

Ahmad Yani,1 Dorothy Dorothy,2 Rizky Amaliah1

1. Department of Surgery, Faculty of Medicine, Universitas Indonesia, Dr. Cipto Mangunkusumo General Hospital, Jakarta, Indonesia
2. Department of Surgery, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
 

Correspondence to: Dorothy Dorothy,MD; Email: dorothy.md@gmail.com

Received: 9 March 2020; Revised: 31 July 2020; Accepted: 7 September 2020; Available online: 2 November 2020

Abstract

Background: In intestinal ischemia, reperfusion towards the injured intestine can cause further injury to the intestine itself and to remote organs. This research aimed to evaluate the influence of intestinal strangulation release (SR) before resection on the intestine outside margin of the strangulated intestine compared with subjects without intestinal strangulation release (WSR). Methods: Fourteen male Sprague Dawley rats were subjected to strangulation of one loop of the distal ileum for 4 h. In the SR group, the strangulated intestine was released for 5 min and then resected for necrotic parts. In the WSR group, the strangulated intestine was immediately resected WSR. The control group received a sham laparotomy. Four hours after the second laparotomy, the animals were sacrificed, and intestinal samples were taken for histomorphological analysis and measurement of intestinal malondialdehyde (MDA) level. Results: The injury on the histomorphological intestinal mucosa and intestinal MDA level were insignificantly higher in the SR group than in the WSR group (p>0.05). Conclusion: Intestinal SR before resection causes more tissue injury and oxidative stress on the intestine outside the strangulation section, but the difference is not statistically significant.

Keywords: Ischemia-reperfusion injury, Intestinal ischemia, Intestinal strangulation release, Malondialdehyde, Intestine injury
Ann Afr Surg. 2021; 18(2): 90-95
DOI: http://dx.doi.org/10.4314/aas.v18i2.6
Conflicts of Interest: None
Funding: None
© 2021 Author. This work is licensed under the Creative Commons Attribution 4. 0 International License.

Introduction

Intestinal strangulation is one of the most common surgical cases that can interfere with the flow of blood vessels to the intestine, as in the case of hernia, volvulus, and intussusception, which also causes half of all cases of death involving small intestine obstruction (1,2). Intestinal strangulation along with its complications has a morbidity rate of 40% (3). Ischemia-reperfusion (I/R) injuries can affect many organs, but the intestine is one of the organs most susceptible to this condition (4). Disorders of blood flow to the intestine can cause ischemic injury, which causes tissue injury, but a more complicated condition can happen when blood flow is restored to the previously ischemic tissue to maintain cell function, known as reperfusion injury. Injuries due to reperfusion often outweigh injuries caused by the previous ischemia (1,4,5).
When the ischemic condition occurs, hypoxanthine, a byproduct of adenosine triphosphate, accumulates. When tissue reperfusion occurs, hypoxanthine and oxygen produce a superoxide which causes inflammatory responses and tissue injury (1,6). In intestinal ischemia with clear boundaries, a release is often performed to assess doubtful intestinal viability. However, the act of strangulation release (SR) can cause reperfusion injury, which may be prevented by resecting all the strangulated part without releasing the strangulation. Knowledge of the effects of intestinal SR is expected to be a consideration for deciding whether SR should be carried out before resecting the ischemic intestine. The best parameters for assessing intestinal injury without clinical signs are histopathological examination and malondialdehyde (MDA) levels.
This research aimed to determine differences in intestinal histomorphological changes and intestinal MDA levels in locations adjacent to strangulation, between intestines that are resected with and without previous SR.

 

 

Material and Methods

Animals
This research was experimental and used male Sprague Dawley rats. The subjects selected were of male sex, healthy, aged between 8 and 12 weeks, with a median bodyweight (BW) of 183.50 (174–213) g. The subjects were received and raised in Pusat Penelitian dan Pengembangan Kesehatan (Puslitbang), Health Research and Development Agency, Ministry of Health of the Republic of Indonesia. This research was approved by the Ethics Committee of Penelitian Kesehatan Fakultas Kedokteran Universitas Indonesia No. 0554/UN2.F1/ETIK/2018 and followed the ethical guidelines for animal research. The sample size in this research was calculated using Federer’s formula approach and considered a 10% dropout correction.
The subjects were given ad libitum food and drink before treatments, their general condition was assessed, and they were weighed to get animals of the same sample size. The subjects were raised in plastic cages 50 × 35 × 15 cm with a battery system base. The cages were kept at room temperature, with indirect sunlight at the same cycle as in nature.

 

Experimental procedures
This research included 14 subjects: 2 rats in the control group, 6 rats in the group that received resection after SR, and 6 rats in the group that received WSR.
At the time of the experiment, the subjects were anesthetized with a 50 mg/kg BW dose of ketamine and 7 mg/kg BW dose of xylazine by intramuscular injection.
A median laparotomy incision of 3 cm was performed along the abdomen. In the two treatment groups, a 10-cm length loop of distal ileum was strangulated using a 2.0 silk ligation, and then the abdomen was closed.
The distal ileum was chosen in this study as it is considered one of the commonest locations of strangulations, especially those caused by intussusception (7). Specifically, in the small intestine, reperfusion after ischemia initiates systemic inflammatory responses which lead to cell and tissue injuries (8). Hence, I/R injury in the small intestine is considered an important clinical problem leading to high mortality and morbidity. The total length of small intestine observed in a subject in this study was 100 cm. This was approximated from a previous study which stated that a rat weighing 125 g has a 95-cm long small intestine, which increases by approximately 5 cm for each 100 g of BW increment (9). Hence, the ratio of strangulated gut versus total gut length in this study was 1:10.
Ligation was released after 4 h of strangulation. Reperfusion duration was determined to be 15 min. This reperfusion time was chosen to resemble the reality of a clinical setting where 15 min is the maximum time allocated to evaluate the intestine’s viability. Thereafter the necrotic intestine was resected after evaluating the intestinal viability in the SR group, and the abdomen was closed. Meanwhile, in the WSR group, the ischemic intestine was resected without releasing the ligation.
Four hours later, two intestinal samples from each rat were taken using a surgical procedure. All SR samples (sample I) were fixed in 10% formalin buffer for histomorphological examination. All WSR samples (sample II) were put into containers that contained 0.9% sodium chloride (NaCl) and stored at 20ºC to measure tissue MDA level. The subjects were then sacrificed by extracting all the blood directly from their hearts. MDA is used as an indicator because MDA is the final product of oxidative decomposition, which is initiated by radicals from polyunsaturated fatty acids (10,11).

 

Histopathological examination of the intestine
A paraffin block was made from sample I; it