Sleeve Gastrectomy in Mice Using Disposable Ligature Clips

Summary

Here, we present a protocol that utilizes a disposable ligature clip to create a mouse sleeve gastrectomy model. This model demonstrates low postoperative mortality, shorter operative time, and therapeutic outcomes comparable to traditional suturing techniques.

Abstract

Current mouse models of sleeve gastrectomy (SG) rely heavily on manual suturing -- a technically demanding procedure with prolonged operative times, high perioperative mortality, and a steep learning curve. This limits their accessibility to non-surgical researchers. To address this bottleneck, we developed a simplified SG protocol using disposable ligature clips, commonly employed in clinical gastrointestinal surgery. In diet-induced obese C57BL/6J mice, the clip-based approach reduced mean operative time by >50% (20.9 ± 3.2 vs. 43.9 ± 3.0 min) and significantly improved 12-week survival compared to traditional suturing. The method achieved equivalent metabolic outcomes, including sustained weight loss, improved glucose tolerance, enhanced insulin sensitivity, and reduced hepatic steatosis. Notably, the model requires no specialized microsurgical expertise or stapling devices, which are often unavailable for murine applications. We recommend this clip-based method for investigators, particularly those without advanced surgical training, who prioritize procedural efficiency and reproducibility in mechanistic studies of bariatric surgery. For surgeons seeking direct clinical translatability, suture-based techniques remain preferable, albeit with more stringent perioperative management.

Introduction

Bariatric surgery is widely acknowledged as the most effective intervention for extreme obesity and its associated complications¹. The mouse sleeve gastrectomy (SG) model is a valuable tool for studying the pathophysiological changes and potential mechanisms associated with bariatric surgery. This model is advantageous because it allows the use of genetically modified mouse strains, such as those with gene knockouts or overexpression. Mouse models that are easy to replicate, enable strict control of experimental conditions, and facilitate experimental manipulation are essential for clinical translation².

The conventional murine SG model utilizes suture-based techniques to fabricate a tubular gastric pouch. This methodology entails multiple precise surgical steps and typically requires substantial technical proficiency. Representative techniques include SG-single-layer^3,4 and SG-Lembert^5,6, with an average surgical time of approximately 30 to 45 min⁷. Consequently, performing SG in a cohort of ten mice generally requires 5-7.5 h of operative time. In studies involving multiple experimental cohorts, cumulative surgical sessions may extend across 2-3 consecutive days. Prolonged procedural timelines can introduce logistical constraints and variability in perioperative conditions, which affect experimental consistency and postoperative monitoring. Researchers have explored alternative approaches to develop more accessible mouse-SG models. Stapler-assisted (SG-stapler)^7,8 and clip-based (SG-clip)^9,10,11 approaches represent notable refinements. These methods streamline specific aspects of gastric pouch formation and reduce the technical demands associated with manual suturing, thereby supporting broader adoption across research settings with varying levels of surgical experience. The SG-stapler technique faces practical limitations in murine applications due to the limited availability of appropriately sized stapling devices, leading to its more frequent use in rat models. Regarding the SG-clip approach, prior studies commonly employed one to two titanium clips supplemented with adjunctive sutures^9,10,11. The smooth inner surface of standard titanium clips provides suboptimal tissue apposition, necessitating supplementary fixation to ensure procedural reliability and gastric integrity.

Drawing on the features of the SG-stapler and SG-clip models, this protocol presents a murine SG model developed using disposable ligating clips. These clips are commonly employed in cholecystectomy, appendectomy, and other gastrointestinal surgeries. In these procedures, they securely clamp tissues such as the residual cystic duct, appendiceal root, and blood vessels, eliminating the need for additional suture reinforcement. Sizes are available that are suitable for the length of a mouse's stomach. This new SG-clip model is straightforward, reliable, and demonstrates therapeutic efficacy comparable to that of the traditional suture model.

Notably, suture-based SG remains preferable when the research aims to replicate clinical surgical technique, investigate suture-specific biomechanical or biological effects, or requires maximal anatomical precision. For investigators with advanced microsurgical skills, hand-sew methods mirror human procedures and are thus more appropriate for translational fidelity. In contrast, the ligating clip model offers a streamlined, efficient, and accessible alternative that maintains experimental rigor while broadening the applicability of murine SG across diverse research settings -- particularly when surgical expertise is limited.

Protocol

This protocol adheres to the International Animal Welfare Ethics Guidelines and has been approved by the Jinan University Experimental Animal Welfare and Ethics Committee (Approval Number: 20240227-0073).

NOTE: Thirty male C57BL/6J wild-type mice (eight weeks old), weighing 22-25 g, were housed in a specific pathogen-free (SPF) environment with a room temperature of 20-26 °C and 12/12 h alternating day/night. After acclimation to a high-fat diet for 4 weeks, the mice were randomly assigned to SC (SG with clip), SS (SG with suture), and SH (sham) groups with 10 mice in each group.

1. Preoperative preparation

1. Change to a liquid diet for 2 days and do not fast before surgery.
   NOTE: Preoperative transition to a liquid diet can facilitate the emptying of solid food from the stomachs of mice, thereby reducing intraoperative contamination. Additionally, it promotes better adaptation to postoperative liquid nutrition, ensuring adequate energy intake.
2. Fix the heating pad on the operating table equipped with an anesthesia mask and adjust the temperature to ~37 °C.
3. Clean the table with 75% alcohol.
4. Extract 1 mL from 100 mL of normal saline to dissolve the penicillin powder. Then, inject the entire dissolved penicillin solution into the remaining 99 mL of normal saline to prepare a penicillin solution with a concentration of 800,000 IU/100 mL.
5. Anesthetize the mice with 3% isoflurane and 0.5 l/min oxygen for approximately 30 s and then place the head into an anesthesia mask with the isoflurane concentration maintained at 1.5%.
6. Sterilize chest and abdominal skin with Anerdian solution and cover with a sterile towel.

2. Surgical procedures

1. Median laparotomy: Lift the skin about 2 cm below the xiphoid process and make a 1.5 cm incision along the midline. Cut open the muscle-free area along the linea alba just enough to fit the retractor (approximately 1.5 cm).
2. Stomach externalization: Using a wet cotton swab, push the colon and part of the small intestine towards the pelvic cavity. Gently pick up the stomach to the abdominal incision with blunt-tipped tweezers and cover it with a new hole towel cut from wet gauze. Carefully dissect the hepatogastric ligament, gastrosplenic ligament, and partial vascular connection of the gastric sinus to the pancreas and cut with an electrocoagulator.
   NOTE: Timely placement of additional new drapes or moistened cotton balls during gastric externalization and incision can effectively reduce contamination from gastric contents.
3. Determine the extent of resection along the greater curvature of the stomach, with the fundus 3 mm distal to the esophagus and the antrum 3 mm proximal to the pylorus.
4. For the SC group:
   1. Place the disposable ligature clip with an applicator along the predetermined extent of the resection; carefully re-determine the distance of the clamp position from the esophagus and the pylorus, then clamp.
      NOTE: Before clipping, the resection line must be confirmed collaboratively by both the assistant and the operator to ensure precise resection margins.
   2. Pre-position a moistened cotton ball around the stomach to prevent contamination of the abdominal cavity with spilling gastric contents; then rapidly cut away approximately 80% of the gastric tissue on the greater curvature side of the ligature clip.
   3. Sterilize the incision three times with Anerdian swabs and wipe once with a saline swab (Figure 1).
5. For the SS group:
   1. Clamp the gastric body along the predetermined extent of the resection.
   2. Pre-position a moistened cotton ball around the stomach to prevent contamination of the abdominal cavity with spilling gastric contents; then use ophthalmic scissors to rapidly cut away approximately 80% of the gastric tissue on the greater curvature side.
   3. Open the forceps and clean the gastric contents with saline swabs.
   4. Suture the incision twice using 6-0 absorbable surgical sutures with a single-layer continuous technique. Perform the first continuous suture by inserting the needle perpendicular to the incision edge, at a distance of 2-3 mm, passing through the full thickness of the gastric wall, and exiting at an equidistant point on the contralateral side. Secure the initial stitch with a surgical knot, continue the suture using alternating 2 mm bites under uniform tension to prevent tissue puckering or gaps, and terminate with a square knot (3-4 throws).
   5. Perform the second continuous suture parallel to the first using an offset pattern to enhance coverage and maintain consistent bite spacing and tension while avoiding overlap or torsion.
      NOTE: Reddening of cotton balls or gauze indicates significant bleeding, particularly at the gastric incision site; adequate electrocoagulation of the incision is required prior to suturing to achieve hemostasis.
   6. Sterilize the incision three times with Anerdian swabs and wipe once with a saline swab (Figure 2).
6. For the SH group:
   1. Perform only the clamping operation of the stomach and release after 10 min.
   2. Return the stomach to the abdominal cavity in the correct position and suture the muscularis propria and skin with 5-0 surgical sutures, sterilize, and end the procedure.
      NOTE: All mice were operated on within one week to facilitate effective monitoring of postoperative weight and food intake.

3. Postoperative care

1. Place the mice in an induction box of pure oxygen. They can be awakened after about 2 min. Remove the mice and place them on a heating pad. Closely observe the state of consciousness and respiration, then return them to the cage when they can move around.
2. Administer a single subcutaneous injection of glucose saline solution (5% glucose and 4.5% sodium chloride) at a dose of 20 mL/kg.
3. Administer a subcutaneous injection of meloxicam at a dosage of 4 mg/kg (concentration: 0.4 mg/mL) immediately prior to the mice regaining consciousness following surgery. Repeat this injection every 24 h for three consecutive days.
4. Provide Abbott nutritional solution and a high-fat diet for the initial three days following surgery; substitute the nutritional solution with drinking water on the fourth postoperative day.
5. Give penicillin (30,000 U/kg) intramuscularly for 3 consecutive days postoperatively to prevent infection.
6. Observe mice daily for general conditions and provide prompt symptomatic treatment as needed.

4. Detection indicators

NOTE: Survival for 4 weeks without significant postoperative complications in mice is considered successful surgery.

1. Record the weight and duration of the surgery at the time of operation, and monitor the weight, food intake, mortality rate, and occurrence of severe complications every week for each group for 12 weeks postoperatively.
2. Perform metabolic and intestinal transit assessments such as intraperitoneal glucose tolerance test (ipGTT), insulin tolerance test (ITT), and small bowel transit test at week 12.
   1. For ipGTT: Fast mice for 12 h. Clip a 1 mm segment from the tail tip and measure fasting blood glucose (time 0). Administer a 20% glucose solution intraperitoneally at a dose of 2 g/kg body weight. Measure blood glucose levels at 15, 30, 60, 90, and 120 min post-administration.
   2. For ITT: Fast mice for 6 h. Measure fasting blood glucose levels. Administer insulin solution (0.075 U/mL) via intraperitoneal injection at a dose of 0.5 U/kg body weight. Measure blood glucose levels at 15, 30, 60, 90, and 120 min post-injection.
   3. For small bowel transit: Fast mice for 12 h. Administer 10% carbon powder suspension via oral gavage at a dose of 10 mL/kg body weight. After 30 min, euthanize the mice. Excise the intact small intestine from the pylorus to the proximal end of the cecum. Measure the intestinal transit ratio as the distance from the pylorus to the leading edge of the carbon suspension divided by the total small intestine length.
3. Sacrifice the mice to observe the anatomical structure of the sleeve stomach and the morphology of the small intestine.
4. Stain the liver samples with hematoxylin-eosin (HE) staining and oil red O staining, and collect the blood samples for the detection of serum total cholesterol (TC), triglycerides (TG), and free fatty acids (FFA).

5. Statistical Analysis

1. Express all continuous variables (body weight, food intake, small intestinal transit ratio, blood glucose levels, and serologic parameters) as mean ± standard deviation.
2. Analyze longitudinal body weight and food intake data using two-way repeated measures ANOVA (factors: group and time).
3. Analyze small intestinal transit ratios and serologic parameters using one-way ANOVA.
4. For intraperitoneal glucose tolerance test (ipGTT) and insulin tolerance test (ITT) data, calculate the area under the curve (AUC) for each subject and compare group AUC values using one-way ANOVA.
5. Perform post-hoc pairwise comparisons with Bonferroni correction following significant ANOVA results.
6. Define statistical significance as p < 0.05. Conduct all statistical analyses using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

Results

The criterion for surgical success was defined as mice surviving for more than 4 weeks post-surgery without experiencing serious complications. Deaths that occurred after the fourth postoperative week are attributed to complications. During 12 consecutive weeks of postoperative observation, one death was recorded in the SC group on the 24th postoperative day due to wound and abdominal infection caused by sutures, while the remaining nine mice survived until the 12th week. In the SS group, a total of five mice died, with ...

Discussion

A murine SG model was established using disposable ligature clips, resulting in a straightforward, efficient, and reproducible surgical procedure. The clip-based technique consistently demonstrated therapeutic efficacy comparable to that of the conventional suture-based SG model, while significantly reducing operative time and improving postoperative survival rates.

The operative technique for the mouse SG-suture model is not inherently complex, and many surgeons experienced in vascular suturi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anerdian solution	Prosperich	HC-3566	Sterilize chest and abdominal skin
75% alcohol	Macklin	E885996-500ml	Clean the surgical table
Animal anesthesia machine	YuyanBio	Yuyan-ABS	Mouse anesthesia equipment
Applicator	HangzhouKangji	Φ10 mm × 330 mm	Disposable ligature clip applicator
Disposable ligature clip	HangzhouKangji	KJ-JZJ02XL	Create a gastric sleeve
Electrosurgical pencil	YuyanBio	Gemini (battery-powered)	Electrocoagulation hemostasis during the operation.
Ensure	Abbott Laboratories	H20181147	Liquid diet
Glucometer and test strips	ACCU-CHEK	Guide Me	Blood glucose monitoring
GraphPad software	San Diego, CA, USA	GraphPad Prism 9	Data statistics and analysis
Heating pad	RTtech	RT-jr01	Maintain a warm body temperature for the mouse during surgery
High-fat diet	Research Diets, Inc	D12492	60 kcal% fat
Isoflurane	RWD	R510-22	Anesthetic drugs
Meloxicam	Aladdin	M129228-25g	Postoperative analgesic drugs
Microsurgical instruments	Beyotime	FS500	Perform animal surgery
Mouse retractor	CHIU POK	CP-ckq	Abdominal surgical retraction in mice
Surgical sutures	Jinhuan Medical	CR537/KCR631	Surgical sutures