Effect of intersphincteric fistula tract ligation versus anal fistulectomy on pain scores and serum levels of vascular endothelial growth factor and interleukin-2 in patients with simple anal fistulas.

OBJECTIVE: This study was performed to explore the effects of ligation of the intersphincteric fistula tract (LIFT) on pain scores and serum levels of vascular endothelial growth factor (VEGF) and interleukin (IL)-2 in patients with simple anal fistulas.
METHODS: Ninety patients with simple anal fistulas were evenly randomized into a study group (treated with LIFT) and a control group (treated with traditional anal fistulectomy) according to a random number table. The surgical outcomes, basic operation conditions (operation time, hospital stay, and anal continence), and postoperative wound healing rates were compared between the two groups.
RESULTS: The study group had significantly better operation conditions (better anal continence and shorter length of hospital stay), a higher postoperative wound healing rate, lower pain scores, higher VEGF and IL-2 levels, and higher overall efficacy rate than the control group. However, the incidence of postoperative complications was not significantly different between the two groups.
CONCLUSIONS: Patients who underwent LIFT had better surgical outcomes, higher wound healing rates, better anal continence, a shorter length of hospital stay, and less severe postoperative pain than those who underwent simple anal fistulectomy. Increased levels of VEGF and IL-2 after surgery may promote wound healing.

RCT Entities:   Population Interventions Outcomes

Introduction

A simple anal fistula is a clinically common surgical disease.[1] It is defined as a pathological connection between the anus and skin and is mainly caused by bacterial infection of the crypt glands in the anus.[2] The main treatment for most fistulas is a traditional surgical procedure in which the fistula is opened; however, this places patients at high risk of postoperative fecal incontinence due to the incision in the anal sphincter.[3] Therefore, new therapies for this condition are needed.

Ligation of the intersphincteric fistula tract (LIFT) is a sphincter-preserving procedure commonly performed in patients with anal fistulas. The internal and external sphincter muscles are separated by blunt dissection in the intersphincteric plane to identify the fistula tract, and the fistula tract is then ligated close to the internal sphincter muscles.[4,5] LIFT is commonly performed for treatment of anal fistulas in the clinical setting.[6] Vascular endothelial growth factor (VEGF) and its receptor are required to balance the formation of new blood vessels and to maintain and remodel the existing vessels within tissues during both childhood development and adulthood.[7] VEGF and its receptor are the major regulators of vascular development and blood and lymphatic vessel function in the human body in both health and disease states.[8] Interleukin (IL)-2, a key cytokine with multiple effects on immune system diseases,[9] has been found to have many prospects for disease treatment. It was one of the first cytokines developed via molecular cloning and is a growth factor required for T-cell proliferation, which is involved in the production of effector and memory cells.[10] Some studies have shown that anal fistulas have different epithelia and are surrounded by dense collagen tissues with pockets of inflammatory cells.[11] High rates of both epithelial-to-mesenchymal transition and transition of proinflammatory cytokines into mesenchymal cells occur in conditions such as cryptorchidism and perianal fistulas, indicating that molecular mechanisms play a role in fistula development and persistence.

Of the studies that have focused on the treatment of simple anal fistulas with LIFT to date, only a few have addressed the roles of VEGF and IL-2.[12] Thus, the present study was conducted to provide a reference for the treatment of simple anal fistulas and assess the effect of LIFT on the VEGF and IL-2 levels.

Materials and methods

Patients’ general characteristics

This randomized controlled trial involved patients with simple anal fistulas who were admitted to our hospital from September 2016 to July 2017. The patients were divided into a study group and a control group according to a random number table. Patients in the study group were treated by LIFT, while those in the control group were treated by anal fistulectomy.

Inclusion and exclusion criteria

The inclusion criteria were a diagnosis of anal fistula via endoanal ultrasound[13]; complete clinical data; no surgical contraindications; no history of surgery or treatment with specialized instruments within the last 6 months; no use of antibiotics within the last 2 months; and the presence of clinical symptoms such as fatigue, fever, listlessness, and chills. The exclusion criteria were perianal dermatitis, lower gastrointestinal tract cancer, inflammatory bowel disease, severe cardiopulmonary insufficiency, severe hepatic or renal dysfunction, combined blood system diseases, and mental illness or a family history of such illness.

This study was approved by the ethics committee of Dongying People’s Hospital, and all participants or their families provided informed consent after they had received an explanation of the study.

Treatment

Endoanal ultrasound and rectal palpation were used to determine the location of the fistula in both groups before surgery. Patients in both groups were anesthetized in the lumbar region with a No. 7 lumbar puncture needle. Lumbar puncture was performed in the L3–4 vertebral space, and 0.75% bupivacaine was injected into the subarachnoid space at a speed of 0.1 mL/s. A 3.0-cm indwelling epidural catheter was inserted at the tail end, and the lumbar puncture needle was pulled out.

In the study group, LIFT was performed as follows. All patients underwent routine skin preparation before surgery. Under lumbar anesthesia, the patient was placed in the lithotomy position to fully expose the lesion and locate the external opening of the fistula. A probe was slowly inserted into the external opening and passed out of the internal opening. The intersphincteric groove was cut at the location of the intersphincteric fistula, and an approximately 1-cm arc-shaped incision was made to separate the tissue downward and thus expose the fistula. The probe was withdrawn, and the fistula was cut off at the lower edge of the fistula into the internal sphincter. The resected end of the proximal internal sphincter was then sutured with 3-0 absorbable suture. A circular incision was made along the edge of the external opening and surrounding tissues were separated using an electrosurgical knife. The fistula was removed along the direction in which it coursed, and the skin and normal subcutaneous tissue above the fistula were preserved. The fistula was removed to the position of the intersphincteric groove. The intact separated fistula was pulled out from the external opening. Wound hemostasis was conducted, and the wound was washed. Oil emulsion gauze was used to fill the wound, and the operation was completed. After the operation, the patients were instructed to maintain a semi-fluid diet and normal defecation for 2 days. The patients were treated with intravenous antibiotics for 3 days to prevent wound infection. The dressing was changed daily beginning the day after the operation as well as after defecation to keep the anus clean. Sitting baths were prohibited for 1 week, after which time the patients were treated with a potassium permanganate sitting bath twice a day.

In the control group, anal fistulectomy was performed as follows. The direction and depth of the fistula were examined with a probe or methylene blue test, and a grooved probe was then inserted through the external opening of the fistula and passed out of the internal opening. A full-thickness incision of the fistula was then made with an electrosurgical knife. If the grooved probe met significant resistance during the penetration process, the tissue was not forcibly penetrated. The covered skin, subcutaneous tissue, and part of the fistula were then cut with an electric cutter along the penetration direction of the grooved probe, and the probe was gradually advanced to penetrate the internal opening of the fistula. After the fistula was completely cut off, the fistula wall, internal opening, and scar tissue around the fistula were removed by the electrosurgical knife. The internal mucosa was sutured, hemostasis and debridement were performed, and sterile gauze was used to pack the wound. The patients were instructed to control defecation within 24 hours after the operation, take a daily sitting bath and clean the surgical wound with benzalkonium chloride solution, change the dressings regularly, keep the wound as clean and dry as possible, and take prophylactic antibiotics for 3 to 5 days postoperatively.

Outcomes

The operation time, length of postoperative hospital stay, and anal continence were assessed and recorded in both groups. The Wexner score was used to assess anal incontinence, with a total score of 20 points among 5 items. Lower scores indicate stronger anal continence ability. Other parameters evaluated were wound exudation, hemorrhage, and the wound healing time. Pain was evaluated using a visual analog scale[14] in the form of a 10-cm slidable vernier caliper anchored by 10 points that define the bounds of various pain dimensions. Higher scores indicate more severe pain. All patients’ pain scores were evaluated on postoperative days 1, 3, and 5. Postoperative complications were also assessed and recorded.

Efficacy of the procedure was evaluated according to the following criteria.[15] Recovery: complete disappearance of all clinical signs and symptoms with good wound healing. High efficacy: elimination of clinical symptoms with slow wound healing. Good efficacy: improvement of clinical signs and symptoms but poor wound healing. No efficacy: no improvement in clinical signs and symptoms, no wound healing, and worsening of condition. The total efficacy rate was calculated as follows: [(recovery + high efficacy + good efficacy)/total number of cases] × 100%.

Assay method of main indicators

Preoperative and postoperative 5-mL fasting blood samples were collected from all participants. The blood was centrifuged at 5000 rpm for 15 minutes to separate serum and red blood cells, and the supernatant was stored at −80°C according to the instructions of the enzyme-linked immunosorbent assay kits for VEGF [item no. JP27101; Tecan (Shanghai) Trading Co., Ltd., Shanghai, China] and IL-2 (item no. C013; Xiamen Huijia Biotechnology Co., Ltd., Xiamen, China). The kit and reagent samples to be assayed were removed from the refrigerator 30 minutes prior to the assay to rewarm them to room temperature. After the blank, standard, and sample wells were set, the standard reagents labeled “SO” with a concentration of 0 were first transferred into the blank wells, and 50 μL of standard reagents in different concentrations were then added into the standard wells. The sample wells were first infused with 10 μL of sample to be assayed and then with 40 μL of diluted sample, both of which were kept away from the blank wells. Next, 100 μL of horseradish peroxidase-labeled detection antibodies were poured into all wells except the blank wells. The reaction well was sealed with a sealing plate and incubated at 37°C for 65 minutes in a water bath. The liquid was then discarded, the wells were filled with washing solution, and each well was dried with an absorbent paper and allowed to stand for 2 minutes. The washing solution was then removed, and the wells were dried with an absorbent paper. This procedure was repeated six times. Next, 50 µL of Substrate A and 50 µL of Substrate B were added to each well, and the wells were incubated at 37°C for 10 minutes in the dark. A fully automated chemiluminescence immunoassay analyzer (Diamond; Beijing Qinye Yongwei Technology Co., Ltd., Beijing, China) was used to measure the optical density of each well at 450 nm within 15 minutes when 50 μL of stop buffer was added to each well. Finally, the VEGF and IL-2 levels were determined.

Statistical methods

Statistical analysis was performed using SPSS version 21.0 (IBM Corp., Armonk, NY, USA). The intragroup enumeration data are expressed as number and percentage of patients, and the chi-square test was conducted to compare the enumeration data between groups. The chi-square test with continuity correction was preferred if T < 5. The measurement data are presented as mean ± standard deviation. An independent-samples t-test was used to compare intergroup measurement data, whereas a paired t-test was used to compare between-group measurement data. In addition, data obtained at multiple time points were compared and analyzed via repeated-measures analysis of variance, and the Bonferroni method was adopted for pairwise comparison of data at each time point in the same group. A P value of <0.05 was considered statistically significant.

Results

General data

Ninety patients were included in this study. The study group comprised 27 men and 18 women ranging in age from 27 to 60 years (mean, 43.28 ± 4.30 years). The control group comprised 31 men and 14 women ranging in age from 29 to 61 years (mean, 43.19 ± 4.40 years). The length of the fistula tract ranged from 30 to 42 mm (mean, 33.73 ± 2.2 mm) in the study group and from 32 to 43 mm (mean, 34.01 ± 2.1 mm) in the control group. The two groups did not differ in terms of sex, age, disease course, body mass index, fistula tract length, place of residence, education, nationality, smoking history, drinking history, exercise history, clinical symptoms, constipation, and other clinical baseline data (Table 1).

Table 1.

General data of patients in the two groups.

Group	Study group(n = 45)	Control group(n = 45)	t/χ2	P value
Sex			0.776	0.378
Male	27 (60.00)	31 (68.89)
Female	18 (40.00)	14 (31.11)
Age, years			0.098	0.922
	43.28 ± 4.3	43.19 ± 4.4
Disease course, months			0.086	0.932
	26.43 ± 4.2	26.51 ± 4.6
Body mass index, kg/m2			0.468	0.641
	25.7 ± 3.9	26.1 ± 4.2
Fistula tract length, mm			0.618	0.538
	33.73 ± 2.2	34.01 ± 2.1
Place of residence			0.241	0.624
Rural	33 (73.33)	35 (77.78)
Urban	12 (26.67)	10 (22.22)
Education			0.747	0.688
Primary school or higher	9 (20.00)	7 (15.56)
High school	16 (35.56)	14 (31.11)
University degree or higher	20 (44.44)	24 (53.33)
Nationality			2.182	0.139
Han nationality	18 (40.00)	25 (55.56)
Minorities	27 (60.00)	20 (44.44)
Smoking history			0.178	0.673
Yes	24 (53.33)	22 (48.89)
No	21 (46.67)	23 (51.11)
Drinking history			1.113	0.291
Yes	21 (46.67)	26 (57.78)
No	24 (53.33)	19 (42.22)
Exercise history			0.714	0.398
Yes	19 (42.22)	23 (51.11)
No	26 (57.78)	22 (48.89)
Clinical symptoms			0.444	0.931
Pain	12 (26.67)	10 (22.22)
Lump	11 (24.44)	13 (28.89)
Itching	13 (28.89)	14 (31.11)
Purulent discharge	9 (20.00)	8 (17.78)
Constipation			0.720	0.396
Yes	39 (86.67)	36 (80.00)
No	6 (13.33)	9 (20.00)

Data are presented as n (%) or mean ± standard deviation.

Comparison of basic operation conditions between the two groups

Significant differences were observed in the operation time, length of hospital stay, and anal continence between the two groups, with patients in the study group outperforming those in the control group (P < 0.05) (Table 2).

Table 2.

Comparison of basic operation conditions between the two groups.

Group	Study group	Control group	t-test value	P value
Number of patients	45	45
Operation time, minutes	49.2 ± 7.4	43.4 ± 6.5	3.95	0.002
Length of hospital stay, days	4.5 ± 1.2	6.7 ± 1.3	8.342	<0.001
Anal continence score	2.34 ± 1.04	6.25 ± 1.07	17.58	<0.001

Data are presented as mean ± standard deviation.

Comparison of postoperative wounds between the two groups

Significant differences in the postoperative wound characteristics were found between the two groups, particularly regarding wound exudation, hemorrhage, and the wound healing time, with the study group showing more favorable results than the control group (P < 0.05) (Table 3).

Table 3.

Comparison of postoperative wound characteristics between the two groups.

Group	Study group	Control group	t-test	P value
Number of patients	45	45
Wound exudate (severity)	2.71 ± 0.17	0.09 ± 0.12	84.46	<0.001
Hemorrhage (severity)	0.32 ± 0.11	0.64 ± 0.13	12.61	<0.001
Wound healing time (days)	16.51 ± 2.01	22.50 ± 3.50	9.956	<0.001

Data are presented as mean ± standard deviation.

Comparison of postoperative pain scores between the two groups at different time points

The pain scores at different time points significantly differed between the two groups (P < 0.05) (Table 4 and Figure 1). The study group had lower pain scores than the control group on days 1, 3, and 5 (P < 0.05), although the scores gradually decreased in both groups (P < 0.05).

Table 4.

Comparison of postoperative pain scores between the two groups at different time points.

Time	Study group(n = 45)	Control group(n = 45)	t-test	P value
Day 1	6.72 ± 0.53*	7.01 ± 0.56	2.523	0.013
Day 3	5.66 ± 0.11*	6.01 ± 0.11	15.090	<0.001
Day 5	2.43 ± 0.24*	3.51 ± 0.25	2.119	0.037
F	1923.000	1130.000	–	–
P value	<0.001	<0.001	–	–

Data are presented as mean ± standard deviation.

*P < 0.05 in the study and control groups.

Figure 1.

Comparison of postoperative pain scores between the two groups at different time points. The postoperative pain scores at different time points were significantly different between the two groups (P < 0.05). The study group had lower postoperative pain scores than the control group on days 1, 3, and 5 (P < 0.05), although the scores gradually decreased in both groups (P < 0.05). *P < 0.05 in the study and control groups.

Comparison of preoperative and postoperative VEGF and IL-2 levels between the two groups

Preoperatively, the VEGF and IL-2 levels were not significantly different between the two groups. Postoperatively, however, the levels were elevated in both groups (P < 0.05) and were significantly higher in the study group than in the control group (P < 0.05) (Table 5 and Figure 2).

Table 5.

Comparison of preoperative and postoperative VEGF and IL-2 levels between the two groups.

Group	n	VEGF level (ng/mL)		T value	P value	IL-2 level (ng/mL)		T value	P value
		Preoperative	Postoperative			Preoperative	Postoperative
Study group	45	6.37 ± 1.58	16.91 ± 3.95	16.620	<0.001	1.78 ± 0.45	4.22 ± 1.66	9.517	<0.001
Control group	45	6.74 ± 1.91	14.22 ± 3.72	12.000	<0.001	1.97 ± 0.66	3.66 ± 1.28	7.872	<0.001
T value	–	1.001	3.326	–	–	0.340	1.792	–
P value	–	0.319	0.001	–	–	0.735	0.076	–

Data are presented as mean ± standard deviation.

VEGF, vascular endothelial growth factor; IL-2, interleukin-2.

Figure 2.

Comparison of (a) VEGF and (b) IL-2 levels between the two groups. No significant difference was observed in the VEGF or IL-2 levels between the study and control groups preoperatively. Postoperatively, however, the study group had significantly higher VEGF and IL-2 levels than the control group (P < 0.05), and a significant increase in the levels was found in both groups (P < 0.05). *P < 0.05 postoperatively compared with that preoperatively; #P < 0.05 postoperatively compared with that preoperatively in the control group.

VEGF, vascular endothelial growth factor; IL-2, interleukin-2.

Comparison of surgical outcomes between the two groups

In the study group, 35 (77.78%) patients showed recovery after LIFT. The procedure showed high efficacy in five (11.11%) patients and good efficacy in three (6.67%) patients. However, no efficacy was observed in two (4.44%) patients, leading to an overall efficacy rate of 95.56%. In the control group, 28 (62.22%) patients showed recovery after anal fistulectomy, 7 (15.56%) showed high efficacy, and 2 (4.44%) showed good efficacy. However, no efficacy was observed in eight (17.78%) patients, leading to an overall efficacy rate of 82.22%. Thus, the study group exhibited significantly higher overall efficacy (P < 0.05) (Table 6).

Table 6.

Comparison of operative efficacy between the two groups.

Efficacy	Study group(n = 45)	Control group(n = 45)	T value	P value
Recovery	35 (77.78)	28 (62.22)
High efficacy	5 (11.11)	7 (15.56)	–	–
Good efficacy	3 (6.67)	2 (4.44)	–	–
No efficacy	2 (4.44)	8 (17.78)	–	–
Overall efficacy rate	43 (95.56)	37 (82.22)	4.050	0.044

Data are presented as n (%).

Comparison of postoperative complications between the two groups

In the study group, three (6.67%) patients developed postoperative pain, one (2.22%) developed hemorrhage, and one (2.22%) developed infection; however, urinary retention was not observed. In the control group, 10 (22.22%) patients developed postoperative pain, 3 (6.67%) developed hemorrhage, 2 (4.44%) developed infection, and 2 (4.44%) developed urinary retention. The postoperative pain scores were significantly lower in the study group than in the control group (P < 0.05). However, there were no significant differences in complications such as hemorrhage, infection, and urinary retention between the two groups (Table 7).

Table 7.

Comparison of postoperative adverse reactions between the two groups.

Group	Study group(n = 45)	Control group(n = 45)	T value	P value
Pain	3 (6.67)	10 (22.22)	4.406	0.036
Hemorrhage	1 (2.22)	3 (6.67)	1.047	0.306
Infection	1 (2.22)	2 (4.44)	0.345	0.557
Urinary retention	0 (0.00)	2 (4.44)	2.001	0.157

Data are presented as n (%).

Discussion

An anal fistula is a common anorectal disease[16] characterized by an abnormal passage between the anal canal and the perianal skin.[17] The treatment of anal fistulas is often challenging, and the available surgical methods are associated with a high rate of recurrence. Thus, more effective techniques must be developed to overcome these challenges.[18,19]

In LIFT, the fistula tract is divided and ligated in the intersphincteric space, and the cavity incision is then closed to reduce the risk of anal fistula recurrence. Compared with fistulotomy, LIFT can better ensure the integrity of the internal and external sphincter muscles and has a low risk of fecal incontinence.[20] Chen et al.[21] described LIFT as an effective sphincter-preserving procedure for the treatment of intersphincteric fistulas with long-term outcomes that are acceptable to patients. Khadia et al.[22] concluded that LIFT is associated with a high healing rate and does not cause incontinence. In the present study, better treatment outcomes were observed in the study group than in the control group in terms of the operation time, length of hospital stay, anal continence, and postoperative wounds. LIFT was proven to be superior to anal fistulectomy in terms of patient recovery, wound healing, and anal continence, thus providing a better healing window. The study group had lower pain scores than the control group on postoperative days 1, 3, and 5, which can be attributed to the fact that LIFT was performed as close as possible to the external sphincter muscle, thereby preventing damage to the internal sphincter muscle and anal mucosa. Moreover, LIFT was effective in suppressing aggravation of the condition, alleviating pain, and promoting recovery. The study group exhibited a higher overall efficacy rate than the control group, indicating better outcomes with LIFT than with anal fistulectomy. Additionally, lower pain scores were observed in the study group than in the control group. However, no significant differences were observed in terms of complications such as hemorrhage, infection, and urinary retention. Thus, compared with traditional anal fistulectomy, LIFT not only has a better effect but also reduces the incidence of postoperative complications, surgical trauma, and postoperative pain.

VEGF is a vascular growth factor[23] that regulates processes such as endothelial cell proliferation, migration, and survival.[24,25] IL-2 is a cytokine that stimulates the expansion of effector T cells and helps T cells to kill cancer cells and virus-infected cells.[26] Kim et al.[27] found that the levels of natural killer cell cytotoxicity and IL-2 in patients with colorectal cancer were significantly increased after surgery, and the increase in both of these parameters improved the patients’ immune function. In addition, Li et al.[28] found that VEGF and basic fibroblast growth factor significantly increased after treatment of anal fistulas, and the increases in these parameters promoted the generation of capillaries, thus improving the blood circulation within the wounds and accelerating wound healing. In the present study, the VEGF and IL-2 levels increased postoperatively in both groups, and the levels were higher in the study group than in the control group. This indicates that LIFT is more effective than traditional anal fistulectomy and accelerates wound healing and that increases in the VEGF and IL-2 levels may help to promote wound healing. This trial was conducted in strict compliance with the inclusion and exclusion criteria to ensure rigor and reliability by eliminating significant differences between the study and control groups in terms of clinical baseline data such as sex, age, and weight. Although LIFT provided better clinical effects than anal fistulectomy, certain improvements are still needed. For example, a comparative study of LIFT and fistulectomy in patients with different disease conditions or different backgrounds might provide useful clinical information. Gradual improvement in research in this field based on the above-mentioned perspectives is expected.

In summary, patients who underwent LIFT had better surgical outcomes, higher wound healing rates, better anal continence, a shorter length of hospital stay, and less severe postoperative pain than those who underwent simple anal fistulectomy. The increased levels of VEGF and IL-2 after surgery may be part of the mechanism that promotes wound healing.