Can olive oil prevent distal colon mucosal atrophy caused by disuse in rats with colostomy? An experimental animal study

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Can olive oil prevent distal colon mucosal atrophy

caused by disuse in rats with colostomy?

An experimental animal study

Czy zastosowanie oliwy z oliwek może zapobiegać

dysfunkcjonalnemu zanikowi dystalnej części okrężnicy u szczurów?

Badanie eksperymentalne na zwierzętach

Ahmet Atıcı

¹

, Emel Demir

²

, Didar Gürsoy

³

, Mehmet Emin Çelikkaya

¹

, Bülent Akçora

¹

1Department of Pediatric Surgery, Mustafa Kemal University, School of Medicine, Antakya, Hatay, Turkey

2Department of Children Health and Diseases Nursing, Mustafa Kemal University, Hatay School of Health, Antakya, Hatay, Turkey

3Department of Patology, Mustafa Kemal University, School of Medicine, Antakya, Hatay, Turkey

Article history: Received: 08.03.2021 Accepted: 05.05.2021 Published: 07.05.2021

ABSTRACT: Aim: This study aims to investigate the effects of pure olive oil on mucosal atrophy of the distal colon in rats.

Material and methods: The study included 28 male albino Wistar rats weighing 300–350 g. A total of 28 animals were randomly allocated to four groups: group 1: control group (n = 6); group 2: ostomy group (n = 6); group 3: ostomy + saline group (n = 8); group 4: ostomy + olive oil group (n = 8). By definition, group 1 did not undergo any procedure. The same surgical procedure was performed for groups 2, 3, and 4 as described below. In all animals from these groups, a two-centimeter-long mid-line incision was made and the colon was divided into 5 cm sections, measured from the distal colon to the caecum.

Proximal and distal stomas were created 2 cm apart in the midline with the use of single-layer interrupted sutures. All rats were kept under close daily observation until they were terminated postoperatively after the 1^st month. Animals from group 2 did not undergo any additional procedures, while those fromgroups 3 and 4 were given 2 mL of saline and olive oil twice a day, respectively. At the end of the 1^st month, the rats were re-operated through the same approach. Biopsies were taken from the proximal and distal stomas of all rats in the same manner for further histopathological analysis.

Results: Group 1 showed no significant differences in terms of mucosal thickness, muscular thickness, wall thickness or colonic lumen diameter between the proximal and distal segments of the colon. Although there were significant differences between the proximal and distal colostomies for each parameter in groups 2, 3, and 4, the mucosal thickness, muscular thickness, wall thickness, and colonic lumen diameter differences for proximal and distal ostomies were very small in group 4 when compared to groups 2 and 3.

Conclusion: The administration of pure olive oil through the distal colon before colostomy closure may reduce the difference in diameter between the proximal and distal intestinal segments. As a result, a more straightforward surgical procedure may be achieved.

KEYWORDS: children, distal colon mucosal atrophy caused by disuse, olive oil

STRESZCZENIE: Cel: Celem niniejszej pracy było zbadanie wpływu czystej oliwy z oliwek na zanik dystalnej części okrężnicy u szczurów.

Materiały i metody: Badanie przeprowadzono na 28 samcach szczurów albinosów rasy Wistar o masie 300–350 g. Wszystkie 28 zwierząt zostało losowo przydzielonych do czterech grup: grupa 1.: grupa kontrolna (n = 6); grupa 2.: osobniki ze stomią (n = 6); grupa 3.: osobniki ze stomią + sól fizjologiczna (n = 8); grupa 4.: osobniki ze stomią + oliwa z oliwek (n = 8). Osobników z grupy 1. z założenia nie poddano żadnym zabiegom medycznym, podczas gdy ten sam zabieg chirurgiczny przeprowadzono u osobników z grup 2., 3. oraz 4., zgodnie z opisem przedstawionym poniżej. U wszystkich zwierząt z tych grup wykonano dwucentymetrowe cięcie w linii środkowej ciała i podzielono okrężnicę na odcinki o długości 5 cm, mierząc je od dystalnej części okrężnicy do kątnicy. Proksymalną i dystalną lufę stomii wyłoniono w odległości 2 cm od siebie w linii środkowej ciała za pomocą zespolenia okrężniczo-mięśniowo-skórnego z użyciem jednej warstwy przerywanych szwów. Wszystkie szczury zostały poddane całodziennej obserwacji do momentu eutanazji po upływie 1 miesiąca od zabiegu. U osobników z grupy 2. nie wykonywano żadnych dodatkowych interwencji. Osobniki z grup 3. i 4. otrzymywały dwa razy dziennie odpowiednio 2 ml soli fizjologicznej lub oliwy z oliwek. Pod koniec pierwszego miesiąca obserwacji, szczury zostały poddane reoperacji w miejscu poprzedniego nacięcia. U wszystkich szczurów pobrano biopsję do badania histopatologicznego z proksymalnej oraz dystalnej części okrężnicy przy użyciu jednolitej techniki.

Wyniki: W grupie 1. nie wykazano istotnych różnic w zakresie: grubości błony śluzowej, grubości warstwy mięśniowej, grubości ściany czy też różnic pomiędzy średnicą światła odcinka proksymalnego i dystalnego okrężnicy. Zaobserwowano jednak istotne

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INTRODUCTION

Colostomy is frequently used in pediatiric contexts for diseases that require multi-stage surgeries, such as anorectal malforma- tion, Hirschsprung disease and colonic atresia [1]. This is especially the case when creation of a diverting ostomy causes lack of exposure to intestinal contents, therefore blocking their trophic effect on intestinal mucosa – in that situation, atrophic changes may occur, affecting the diameter and wall thickness of the distal intestinal segment [2–5]. In some congenital anomalies, such as intestinal atresia, meconium ileus or colon atresia, which require creation of a stoma, significant diameter differences are observed in the proximal and distal intestinal segments. Clini- cal observations confirm proximal and distal intestinal diameter differences in patients suffering from the above-mentioned diseases (especially intestinal atresia, meconium ileus, and colon atresia), which then lead to surgical difficulties during ostomy closure, as opposed to cases without diameter differences. In some previous studies, various substances like short-chain fatty acids (SCFA) have been used to prevent atrophy caused by disuse [1, 6–10]. Our study examines the effect of pure olive oil (Elea olive oil Antioch, Hatay/Turkey, 2020) on distal colon atrophy. Olive oil, which is associated with the Mediterranean diet, is obtained through mechanical extraction of fruit from the Olea europea L.

tree [11]. This study investigates the effects of pure olive oil on mucosal atrophy of distal colon in rats.

MATERIALS AND METHODS

The study was conducted at the Mustafa Kemal University Experi- mental Animal Laboratory, Hatay, Turkey, and was approved by the University’s animal research ethics committee (31/10/2019/08-6).

Male albino Wistar rats (N = 28) weighing 300–350 g were kept in an experimental environment conditions maintained at 20–22°C with approximately 55% humidity and a 12 h day-night cycle during one week of adaptation. All animals (N = 28) were randomly allocated to four groups: group 1: control group (n = 6); group 2: ostomy group (n = 6); group 3: ostomy + saline group (n = 8); group 4: ostomy + olive oil group (n = 8).

Surgical procedure

The surgical procedure and examination under anesthesia before euthanasing the animals were performed with a 2.5 mag- nification loupe (Rose Micro Solutions/West Seneca/USA). All surgical procedures were carried out with antibiotic prophylax- is (ceftriaxone – 100 mg/kg). By definition, animals from group 1 did not undergo any procedures. Experimental surgical procedures described below were performed on subjects from group 2, group 3 and group 4. All subjects were anesthetized with

intramuscular injection of ketamine (50 mg kg⁻¹) and xylazine (10 mg kg⁻¹), preceded by 12 h of fasting. The sterile rats’ abdo- mens were then shaved under anesthesia. A two-centimeter-long mid-line incision was made and the caecum was exposed through this incision. The colon was divided into 5-cm segments, measured from the distal colon to the caecum in all cases. Proximal and distal stomas were created 2 cm apart in the midline with the use of single-layer interrupted sutures (5–0 vicryl). To prevent faecal spillage into the distal segment, a 0.5-cm margin was maintained between the proximal and distal stomas. Next, peri- toneum, muscleand fascia with skin were closed in layers with 4/0 vicryl interrupted sutures (Fig. 1A.). The wound was cleaned with povidone-iodine solution and the animals’ cages were cleaned twice a day for seven days after surgery. All rats were kept under close daily observation until they were terminated postoperatively after the first month. Animals from group 2 (Fig. 1B.) did not undergo any additional interventions. Animals from group 3 (Fig. 1C.) and group 4 (Fig 1D.) were given 2 mL of saline and olive oil twice a day, respectively (saline and olive oil were given via a 5-Fr feeding catheter, see Fig. 1D.). At the end of the first month, the rats were re-operated through the same approach (the first surgical intervention for animals from group 1). Biop- sies from proximal and distal stomas were taken in the same way Fig. 1. (A) Postoperative divergan ostomy in rats. (The yellow arrow shows proximal colon, the black arrow shows distal colon); (B, C, D) Postoperatively after the 1st month of group 2 (B), 3 (C), 4 (D) (The yellow arrow shows proximal colon, the black arrow shows distal colon).

różnice między proksymalnym i dystalnym końcem stomii dla każdego z wymienionych parametrów w grupach: 2., 3., i 4.

W grupie 4. różnice grubości błony śluzowej pomiędzy końcem proksymalnym i dystalnym stomii, jak również różnice grubości mięśni, grubości ściany jelita czy średnicy światła okrężnicy, były bardzo niewielkie w porównaniu z grupami 2. i 3.

Wnioski: Podanie czystej oliwy z oliwek przez dystalną część okrężnicy przed zamknięciem kolostomii może zmniejszyć dysproporcję między średnicą proksymalnego i dystalnego odcinka jelita. Zmniejszenie różnic pomiędzy średnicą bliższego i dalszego odcinka okrężnicy pozwala na uproszczenie zabiegu chirurgicznego.

SŁOWA KLUCZOWE: dzieci, niefunkcjonalny zanik dystalnej części okrężnicy, oliwa z oliwek

A B

D

C

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from all animals. Since animals from group 1 were operated for the first time, biopsies were obtained from proximal and distal segments in the same area where stomas were created in animals from other groups. Tissue samples were placed in a 10% buffered formaldehyde solution for histopathological analysis.

Pathological evaluation

All of the intestinal tissue samples were fixed in a 10% buffered formaldehyde solution for 24 hours. Dehydration of the samples was achieved by passing specimens through a series of alcohol solutions in increasing concentrations. The samples were then cleared with xylene and embedded in paraffin with the use of a closed-system tissue-tracking device (Thermo scıentific, Excel- sior ES) for 14 hours. After routine tracking of tissues embedded in paraffin, a series of 3–5 micrometer-thick sections were cut from each paraffin-embedded block with the use of a rotary micro- tome. The sections were stained with hematoxylin and eosin (H&E) and evaluated using a light microscope (BX51, Olympus, Japan).

For each group, the thickness of mucosa, muscularis propria and thickness of the entire colonic wall were evaluated. The height of each layer was calculated by measuring the distance between two points and an average of 30 measurements were used for each parameter. Photos of slides examined under the BX51 light microscope (Olympus, Japan) were taken with the use of DP20 (Olympus, Japan) image-analysis system attached to the microscope. Colonic lumen widths were measured macroscopically. The examinations were performed by an independent pathologist who was not fa- miliar with the study groups.

Statistical analysis

Data analysis was performed using IBM’s SPSS statistical package (Windows v. 20.0, IBM Corp., Armonk, NY, USA). First, a Shap- iro-Wilk test was used to measure the distribution of the scale variables which turned out to be normal (P > 0.05). Data was analyzed using repeated-measures ANOVA tests and the differences

within each group were analyzed using a paired t-test; values below P < 0.05 were accepted as significant.

RESULTS

According to the results of repeated measures ANOVA test, factors influencing the proximal-distal diameter differences and proximal-distal diameter differences x group factor interactions for all parameters (mucosal thickness, muscular thickness, wall thickness and colonic lumen diameter) were statistically significant (P < 0.05). No statistical difference was found in terms of mean values observed in study groups for all parameters (P > 0.05).

Based on the paired t-test results, the measurements of the macroscopic lumen diameter (cm), microscopic mucosal, muscular and colon wall thickness (micron), and proximal and distal colostomy values for group 1 (Fig. 2A., B.), group 2 (Fig. 2C., D.), group 3 (Fig.

2E., F.), and group 4 (Fig. 2G., H.) are summarized in Tab. I.−IV., respectively. As seen in Tab. I., group 1 showed no significant differences in terms of mucosal thickness, muscular thickness, wall thickness, and colonic lumen diameter between the proximal and distal colon segments (P = 0.772, 0.631, 0.087, and 1, respectively).

Although there were significant differences between the proximal and distal colostomies for each parameter in groups 2, 3, and 4, the mucosal thickness, muscular thickness, wall thickness, and colonic lumen diameter differences for proximal and distal ostomies were very small in group 4 (Fig. 2G., H.) when compared to groups 2 (Fig. 2C., D.) and 3 (Fig. 2E., F.; Tab. II.−IV.). Although one rat from the saline group died during follow-up, no other complica- tions were encountered during the follow-up period.

DISCUSSION

When diverting ostomies are performed in pediatric patients due to congenital anomalies, intestinal contents cannot pass to the Fig. 2. (A., B). Proximal mucosal and muscle thickness in the control group (H+E, x40) (A); distal mucosal and muscle thickness in the control group (H+E, x40) (B) (The red arrow shows mucosal thickness, the blue arrow shows muscle thickness). (D) Proximal mucosal and muscle thickness in the ostomy group (H+E, x40) (C); distal mucosal and muscle thickness in the ostomy group (H+E, x40) (D). (The red arrow shows mucosal thickness, the blue arrow shows muscle thickness). (E, F) Proximal mucosal and muscle thickness in the saline group (H+E, x40) (E); distal mucosal and muscle thickness in the saline group (H+E, x40) (F.). (The red arrow shows mucosal thickness, the blue arrow shows muscle thickness). (G, H) Proximal mucosal and muscle thickness in the olive oil group (H+E, x40) (G); distal mucosal and muscle thickness in the olive oil group (H+E, x40) (H) (Red arrow shows mucosal thickness, blue arrow shows muscle thickness).

A

E F G H

B C D

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surgical procedures e.g. fish-mouth closure. Luminal contents, such as bile, food, pancreatic secretions, and intestinal microbiota are important factors that prevent atrophy of intestinal mucosa [2, 12]. However, poor dietary habits prevent fatty acid formation and absorption in the instestinal lumen, therefore leading to atrophy [2, 8, 10]. Colorectal mucosal epithelial cells use short-chain fatty acids, particularly acetic, propionic and butyric acids, as the primary source of energy [3, 5, 8, 10]. The study investigatsthe effect distal colon. As a consequence, atrophic changes occur in the

muscularand mucous layers of the relevant portions of the distal colon [2, 6, 8, 10]. The presence of atrophic changes within the walls of the distal colon has already been documented in previous studies [1, 2, 6, 8, 10]. According to our clinical observations, especially in certain diseases, atrophy of the intestinal mucosa is observed during ostomy closure. Moreover, if the diameter difference is significant, some surgeons decide to perform additional

PROXIMAL DISTAL

Mean Std. Deviation Mean Std. Deviation P value

Mucosa (micron) 396.4 64.4 395.8 61.3 .772

Muscle (micron) 400.7 106.7 398.9 105.1 .631

Colonic wall thickness (micron) 895.0 150.6 889.5 155.2 .087

Colonic luminal diameter (cm) .31 .075 .31 .09 1.000

Mucosa (micron) 423.7 132.4 322.0 123.6 .001

Muscle (micron) 345.3 141.2 263.9 108.1 .048

Colonic wall thickness (micron) 818.7 317.3 657.7 232.4 0.04

Colonic luminal diameter (cm) .35 .1 .23 .12 0.001

Mucosa (micron) 362.8 118.5 214.5 88.9 .012

Muscle (micron) 454.3 125.8 172.2 103.6 .009

Colonic luminal diameter (cm) .34 .07 .20 .05 .008

Mucosa (micron) 398.8 160.9 334.2 153.4 .026

Muscle (micron) 379.3 135.9 307.3 111.9 .002

Colonic luminal diameter (cm) .40 .18 .30 .13 .033

Tab. I. Macroscopic lumen diameter (cm) and colon wall thickness (micron); microscopic mucosal and muscular thicknesses (micron) with statistically analyzed proximal and distal colon values of group 1.

Tab. II. Macroscopic lumen diameter (cm) and colon wall thickness (micron); microscopic mucosal and muscular thicknesses (micron) with statistically analyzed proximal and distal colon values of group 2.

Tab. III. Macroscopic lumen diameter (cm) and colon wall thickness (micron); microscopic mucosal and muscular thicknesses (micron) with statistically analyzed proximal and distal colon values of group 3.

Tab. IV. Macroscopic lumen diameter (cm) and colon wall thickness (micron); microscopic mucosal and muscular thicknesses (micron) with statistically analyzed proximal and distal colon values of group 4.

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lumen in an antegrade manner, in order to prevent distal colon atrophy – the results showed that distal colon atrophy developed in all groups except for the control group. However, when the proximal and distal intestinal lumen diameters were measured and compared with one another, it turned out that, though there were significant differences between proximal and distal colostomy diameters between group 2 and group 3, this difference was significantly smaller in group 4 when compared to group 2 and group 3 (Tab. II., III., and IV.).

Despite its advantages, we identified two primary limitations of this study: (I) the use of animal subjects and (II) a relatively short follow-up time.

CONCLUSION

Administration of pure olive oil through the distal colon prior to colostomy closure may reduce the difference in diameter between the proximal and distal intestinal segments. As a result, a more straightforward surgical procedure may be achieved. This is especially important for ostomies performed to treat intestinal atresia, meconium ileus and colon atresia, which require more complex surgical procedures. Therefore, further clinical studies are required to investigate whether this method can truly reduce atrophy.

of oleic acid from pure olive oil on patients with distal colostomy.

Olive oil is readily available and relatively inexpensive (in Turkey, the current price is approximately $3.5 US per 1000 mL). There- fore, we believe that olive oil can be easily provided to patients in developing countries. Olive oil is a complex mixture of free fatty acids and triglycerides (98%), and a large number of other minor components such as phenolic compounds and sterols (2%) [11].

Olive oil’s myriad qualities (antioxidant, anti-inflammatory, anti- atherogenic, anti-ulcer, anti-cancer, anti-microbial, anti-diabetic, anti-oxidative effect on brain tissue, skin protection, and anti-ag- ing) have been widely investigated in literature [11, 13, 14]. Sev- eral experimental studies have concluded that a diet rich in extra virgin olive oil could be beneficial for ulcerative colitis [15, 16].

In other studies, SCFA, long-term therapy with biosynthetic human growth hormone, glutamine as rectal enema and intestinal lumen irrigation, administration of fibers, psyllium and Maharishi Amrit Kalash (MAK) have also been used for prevention of atrophic changes in the intestinal mucosa [2, 5, 8, 10]. These studies have shown that, while fibers and glutamine are effective in preventing atrophy, other components are not. In addition, other studies have also shown that SCFA infusion into the colon has a trophic effect on the rectal mucosa [5, 7, 9]. A clinical study conducted by Atıcı et al.

concluded that olive oil reduced mucosal atrophy in the distal colon [1]. In this study, olive oil was administered directly to the distal colon

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Word count: 3157 Page count: 6 Table: 4 Figures: 2 References: 16

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Ahmet Atıcı, Associate Professor; Mustafa Kemal University, School of Medicine, Department of Pediatric Surgery, 31124, Antakya, Hatay, Turkey; Phone: +09 05052583057; E-mail: ahmetatici06@gmail.com, ahmetim1501@hotmail.com Atici A., Demir E., Gursoy D., Celikkaya M.E., Akcora B.: Can olive oil prevent distal colon mucosal atrophy

caused by disuse in rats with colostomy? An experimental animal study; Pol Przegl Chir 2021; 93 (1–6);

DOI: 10.5604/01.3001.0014.8696 (Advanced online publication) DOI:

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