The Anti-Adhesive Effect of Granulocyte-Colony Stimulating Factor on an Abdominal Adhesion Model in Sprague-Dawley Rats

Background: Intra-abdominal adhesions are among the most common complications following abdominal surgery. In this study, using an abdominal adhesion rat model, we investigated the anti-adhesive effect of granulocyte-colony stimulating factor (G-CSF) on intra-abdominal adhesions. Methods: All rats were laparotomized with 3 ischemic peritoneal buttons developed to cause adhesions. The experimental rats were divided randomly into 3 groups (n=8/group): control, pluronic gel, and G-CSF groups. Fourteen days after surgery, all rats were sacriced, and intra-abdominal adhesions were assessed. The percentage of adhesions, adhesion severity scale and density of adhesion formation were evaluated. Real-time PCR was conducted to assess the cytokine mRNA levels of substance P (SP), neurokinin 1 receptor (NK-1R), transforming growth factor-β1 (TGF-β1), and intercellular adhesion molecule-1 (ICAM-1). Results: The severity scores of intra-abdominal adhesions of, and the degree of adhesion formation in, rats treated with G-CSF were signicantly lower than those in case of rats from other groups. Additionally, in the G-CSF group, the number of ischemic buttons with developed adhesions was signicantly lower than that in the other groups. In adhesion samples of the G-CSF group, the expression level of SP was signicantly lower than that in the other groups. Conclusions: Our study demonstrated that G-CSF treatment decreases the formation of intra-abdominal adhesion after surgery by reducing inammatory reactions in adhesion tissues.


Background
Adhesions, which are described as an irregular union of two opposing tissue surface, are a common result of the normal healing reaction. After abdominal surgery, most patients develop intraperitoneal adhesions [1,2]. Intra-abdominal adhesions are an important cause of postoperative morbidity. These adhesions are known to be complications associated with abdominal surgery, and they can cause abdominal pain, bowel obstruction, infertility, and chronic pelvic pain [3].
In ammation is the initial reaction to abdominal trauma during adhesion formation, and it plays an important part in the formation of intra-abdominal adhesions [4]. Fibrin exudation and deposition triggers the development of intra-abdominal adhesions due to an in ammatory reaction in the damaged peritoneum. Moreover, a reduction in the capability of lysis brin in damaged sites can also lead to adhesion [5].
Granulocyte colony-stimulating factor (G-CSF) is a cytokine known for stimulating the mobilization of hematopoietic cells from the bone marrow to the peripheral blood. One function of G-CSF is to stimulate the proliferation and differentiation of neutrophil precursors [6]. Recent studies have shown that G-CSF modulates the expression of speci c anti-in ammatory cytokines. These experiments revealed that G-CSF decreases the levels of pro-in ammatory cytokines by inhibiting the development of in ammatory mediators, including interferon-γ, tumor necrosis factor-α, and interleukin-1 [7,8]. In addition, our previous study reported that the wound healing effect of the local G-CSF injection accelerated the deposition of collagen within the dermis around the wound, resulting in a decreased in ammatory response in wound tissues [9].
Based on reports regarding in ammation, we speculated that G-CSF may have anti-adhesive effects in adhesion formation. We investigated the anti-adhesive effect of G-CSF in a rat model of intra-abdominal adhesion and explored the expression of various in ammatory cytokines in adhesion tissues.

Animals
In this study, 6-week-old male Sprague-Dawley (SD) rats purchased from Koatech Inc., Republic of Korea, weighing 260-280 g were used. The SD rats were kept at the Animal Experiment Center of Hanyang University in a speci c pathogen-free environment. Temperatures were kept at 23-25℃ and humidity was maintained at 50-60% with an arti cial light/dark cycle of 12:12 h. The animal research protocol was approved by the Institutional Animal Care and Use Committee of Hanyang University (HY-IACUC-20-0003), and animal studies were conducted in accordance with the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines [10].

Establishment of the abdominal adhesion model
The abdominal adhesion model was induced in rats using procedures based on previously described methods [11]. The rats anesthetized with a combination of rompun (10 mg/kg, Bayer, Seoul, Korea) and zoletil 50 (30 mg/kg, Virbac SA, Carros, France). All rats underwent ventrotomy through a 3 cm skin incision in the midline of the abdomen. Three ischemic buttons were prepared within the parietal peritoneum by gripping a 5 mm parietal peritoneum button with a mosquito hemostat and connecting the bottom of the segment with a 4-O black silk suture (AILEE Co., Pusan, Korea) as described previously [12].
The rats were divided randomly into three groups. Rats in the control group received no medication (n = 8). Rats in the pluronic gel group (n = 8) received only Pluronic F127 gel on the ischemic buttons, and rats in the G-CSF group (n = 8) received a mixture of Pluronic F127 gel and G-CSF (60 µg, Dong-A, Seoul, Korea) on the ischemic buttons (Fig. 1A). The incision was closed with a 3-O polyglycolic acid suture (AILEE Co.) for the fascia and a continuous 5-O polyglycolic acid suture (AILEE Co.) for the skin. After two weeks, the euthanasia of rats induced by carbon dioxide (CO 2 ) inhalation to obtain samples for molecular analysis and to evaluate and score the adhesion.
Scoring for adhesion At 2 weeks after surgery, all rats were assessed according to the standard adhesion scoring system [12][13][14]. The percentage of adhesions was calculated as the number of adhesions in a rat. The adhesion severity scale was characterized as follows: grade 0 = no adhesions, grade 1 = avascular or thin adhesion, grade 2 = dense or vascularized adhesion, and grade 3 = rm or cohesive attachment. The adhesion density was characterized as follows: grade 0 = no adhesions, grade 1 = adhesion was isolated from tissue without gentle traction, grade 2 = adhesion was isolated with mild traction, and grade 3 = adhesion required dissection as described previously [15]. All rats were evaluated by two observers who were blinded to the animal groups.

Statistical analyses
Statistical analyses were conducted using the Statistical Package for the Social Sciences 24.0 software (IBM Co., Armonk, NY, USA). All data are expressed as the mean ± standard error (SE). The statistical difference between groups was analyzed using one-way analysis of variance for multiple comparisons, and post hoc multiple comparisons were performed with Tukey's test (equal variances assumed). Pvalues less than 0.05 were considered statistically signi cant.

Development of the abdominal adhesion model
The ischemic buttons were generated on the parietal peritoneum on day 0 (Fig. 1). Adhesion in the G-CSF group was con rmed on day 14 after treatment with G-CSF and pluronic gel. The arrows indicate the ischemic buttons with attached adhesions (Fig. 2).

Adhesions severity scale
The adhesion severity scale was assessed by the adhesion scoring system. The severity of adhesions was signi cantly lower in the group of rats treated with G-CSF than that in the untreated group (1.19 ± 0.21% vs. 2.52 ± 0.11%, p < 0.05). In addition, the adhesion severity was signi cantly lower in G-CSFtreated rats than in treated with pluronic gel (1.19 ± 0.21% vs. 1.96 ± 0.13%, p < 0.05) (Fig. 3A).

Grade of adhesion density
The grade of adhesion density was assessed by the adhesion scoring system. This grade was signi cantly lower in the group of rats treated with G-CSF than in the untreated rats (0.95 ± 0.18% vs. 2.43 ± 0.14%, p < 0.05). In addition, the grade of adhesions density was signi cantly lower in treated with G-CSF than in those treated with pluronic gel (0.95 ± 0.18% vs. 2.0 ± 0.22%, p < 0.05) (Fig. 3B).

Percentage of ischemic buttons with adhesion formation
The percentage of adhesion formation was signi cantly lower in the rats treated with G-CSF than in the untreated rats (66.67 ± 12.60% vs. 100.0%, p < 0.05). In addition, the percentage of adhesion formation was signi cantly lower in the rats treated with G-CSF than in the rats treated with pluronic gel (66.67 ± 12.60% vs. 95.24 ± 4.76%, p < 0.05) (Fig. 3C).

Molecular analysis
The expression level of SP was signi cantly lower in adhesion samples from rats treated with G-CSF than in those from untreated rats or rat treated with pluronic gel (0.08 ± 0.02% vs. 0.14 ± 0.03% and 0.16 ± 0.03%, respectively; p < 0.05 for both comparisons). The expression levels of NK-1R, TGF-β1, and ICAM-1 were not signi cantly different among the groups (Fig. 4).

Discussion
In this study, we examined the anti-adhesive effect of G-CSF in an abdominal adhesion rat model, as well as the expression of several in ammatory cytokines in adhesion tissues. Our study showed that G-CSF treatment decreased the severity of adhesions, grade of the adhesion density, and the percentage of adhesion formation in the abdominal adhesion rat model. Additionally, G-CSF treatment reduced SP mRNA expression in the adhesion tissues.
Intra-abdominal adhesion formation following surgery can result in serious complications, including abdominal pain, intestinal obstruction, and possible chronic pelvic pain [3,16]. Various surgical methods have been used to treat abdominal adhesions; adjuvants have also been proposed for treating adhesion formation. The development of surgical techniques may reduce abdominal adhesion, but such techniques cannot eliminate it completely [17][18][19]. Many anti-adhesion agents are used, which can be classi ed as systemic or intraperitoneal agents and intraperitoneal barriers [2,20,21]. However, despite these anti-adhesion agents, many problems arise. The adhesion formation process has been explored in different ways and can be divided into four stages: an in ammatory post-injury stage, a brin dissolving stage, a brous band stage, and a phagocytic and remodeling stage [22][23][24]. Many studies have determined that in ammatory responses play an important role in the formation of adhesions [25,26]. Wei et al. [27] reported that gallic acid reduces adhesions formation in rats by inhibiting in ammatory reactions. Our ndings also showed that the in ammatory cytokine level of SP was decreased by G-CSF treatment. Moreover, adhesion formation has also been found to be reduced, along with a reduction in the in ammatory response. Therefore, the in ammatory response may play a signi cant role in the development of adhesions.
SP is secreted by a variety of in ammatory cells, including lymphocytes, eosinophils, macrophages, and dendritic cells. SP also functions by binding to the NK-1 receptor [28]. Some studies have shown that preventing the pro-in ammatory effects of SP may have potential therapeutic effects in in ammation related diseases [29,30]. Our study con rmed that G-CSF treatment decreases SP mRNA expression in adhesion tissues, which indicated that SP indirectly prevents adhesion formation.
G-CSF is an important regulator of hematopoiesis and the innate immune system; for example, it stimulates the proliferation, survival and differentiation of neutrophil precursors and mature neutrophils and the mobilization of bone marrow cells [31]. However, recent studies shown that G-CSF exerts a local effect on the healing process in damaged tissues [32]. In particular, our previous study showed that the local effect of G-CSF reduces the in ammatory response during the healing process, as well as accelerates this process [9]. Our results also showed that the local actions of G-CSF reduced abdominal adhesions and in ammatory cytokines.
This study has several limitations. First, the multiple mechanisms underlying the anti-adhesive effects of G-CSF on adhesion formation could not be clearly de ned. Second, we did not set an optimal dosage range for G-CSF. Further investigations concerning the appropriate dose of G-CSF are thus required. Finally, although we found that G-CSF reduced abdominal adhesion, we did not compare the results with those of other anti-adhesion agents. Further studies will need be required to compare the effects of G-CSF against those of other anti-adhesion agents.

Conclusion
In conclusion, our research showed that G-CSF treatment reduced the development of postoperative intraabdominal adhesions and SP mRNA expression in adhesion tissues. Anti-in ammatory activity is also a possible mechanism of underlying the action of G-CSF. The anti-adhesive effect of G-CSF is associated with the alleviation of in ammatory reactions, which may play an important role clinically.

Declarations
Ethics and Consent to participate

Consent for publication
Not applicable.

Availability of data and material
The datasets used are available from the corresponding author on reasonable request.