A Modified Technique of Mouse Vertical Sleeve Gastrectomy Using Two Disposable Micro-clamps

Vertical sleeve gastrectomy (VSG) is one of the most common forms of bariatric surgery, and its beneficial effects on morbid obesity and accompanying comorbidities have widely been accepted [1]. Its efficacy and safety have widely been proven, but its underlying mechanisms that lead to weight loss and metabolic improvements are still controversial [2]. Most researchers studying such mechanisms use a mouse model of VSG, and there have been various surgical techniques to perform VSG in mice [3]. However, some techniques required tedious, time-consuminng continuous sutures along the gastric wall before gastrectomy, and some techniques used metal clips which were left permanently in the abdomen [4,5]. To overcome such demerits and to simplify the existing techniques, we describe herein a modified technique of mouse VSG using 2 disposable micro-clamps.


INTRODUCTION VSG in Mice
Prior to surgery, the mouse was weighed to obtain a baseline weight measurement. The surgical room was equipped with a state-of-the-art anesthesia system, heated water bath, and heating pad set to 37°C, to keep the animal warm during surgery and recovery after anesthesia. Warm saline solution (0.9%) was used for irrigation. Prior to any surgical procedures, the surgical field was cleaned with 70% ethanol, and proper surgical techniques were used to open the autoclaved surgery pack and dissecting equipment. The mouse was anesthetized using an induction chamber with 5% isoflurane and an oxygen (O 2 ) flow rate of 1 L/min. The absence of a hind-limb pinch-withdrawal reflex was verified to ensure the proper degree of anesthesia. Following this, the mouse was maintained with 1% to 3% isoflurane and an O 2 flow rate of 0.6 L/min. Eye ointment was placed on the animal's eyes to keep them moist during surgery. An analgesic (buprenorphine 1 mg/kg) was also administered subcutaneously. Hair from the umbilicus to the xyphoid process was shaved. Following shaving, the abdominal skin was prepared with povidone-iodine and 70% alcohol. The surgical field was prepared by placing sterile surgical drapes on either side of the mouse, and autoclaved tin foil was used to create a surgical drape for the mouse abdomen. A small hole was cut in the tin foil to allow access to the abdomen area. An incision in the skin from the mid-abdomen (umbilicus) to the level of the xiphoid cartilage was made using an iris scissors, the linea alba was identified, and a cut was made through the body wall along the linea alba. Cotton tip applicators (CTAs) were used to gently elevate the stomach out of the abdomen and the greater omentum was removed from the greater curvature by blunt dissection. Following this, the short gastric artery that runs between the fundus of the stomach and the spleen was divided with electrocautery. The stomach was then exteriorized from the abdominal cavity, and a gauze pad was placed under the stomach and wet with saline ( Figure 1A). A small gastrotomy incision was made along the greater curvature of the corpus, and the remaining gastric contents were removed by gentle squeezing with the CTAs. For VSG with antrum preservation, the line of transection started from the angle of His and ended at the margin of the pancreas gastric lobe so that at least 80% of the gastric volume could be removed after surgery. Two micro-clamps (TKMV-1, Baxter, IL, United States; one from the angle of His and one from the pancreas margin) were applied along this line, and spring scissors were used to cut the gastric sleeve ( Figure 1B and 1C). For VSG without antrum preservation, the pancreas gastric lobe was resected from the stomach to expose the greater curvature of antrum, and the micro-clamp was applied just beneath the pylorus to remove most of the antrum. The cut edge was closed with 7-0 monofilament nonabsorbable suture with a tapered needle by removing the clamp backward at intervals of 1 mm and then placing simple, interrupted stitches to close the exposed edge (see attached video for details ABSTRACT Vertical sleeve gastrectomy (VSG) is a type of bariatric surgery that is accepted as the simplest but most efficient surgical modality to treat morbid obesity. A mouse model of VSG has been widely utilized to investigate the underlying mechanisms of VSG. There are numerous microsurgical techniques for VSG in mice, and we report herein a modified technique with 2 disposable micro-clamps to simplify the surgery. The key aspects of our modification are to use the micro-clamps as non-crushing tissue clamps for anastomosis as well as vascular clamps for hemostasis. Mean body weight at postoperative week 1 was 24.1 grams, which is equivalent to a 16.1% loss of original body weight, and there was no mortality after antrum preservation VSG. This simplified surgical technique will best serve as a tool to investigate either the underlying mechanisms of VSG or the intrinsic change to the stomach after VSG.
of the procedure at https://doi.org/10.24983/scitemed.imj.2020.00137). An average of 20 knots was used to securely close the stomach, and any gastric remnants were flushed with saline throughout the procedure in order to keep the tissue moist and clean. After an additional lavage, the stomach was placed back into the abdominal cavity under the liver using a CTA. The abdominal muscle layer and skin layers were closed using 6-0 monofilament absorbable suture with a tapered needle in a simple, discontinuous pattern. The mouse was then allowed to recover on a heating pad for 10 to 15 minutes before returning it to its home cage. The animal was continuously attended until it has regained consciousness and was able to move freely.

Postoperative Mouse Care and Measurement
Following the return of the mice to their home cages, the cages were placed on a heating pad. Mice were maintained on a liquid diet for 5 days following surgery. An analgesic buprenorphine (1 mg/kg subcutaneously) was administered for 2 days after surgery. During the postoperative period, the body weight of animals was accessed on a daily basis until postoperative 7 days, and then at 2 weeks and 4 weeks thereafter. Food intake, defecation, activity level, and disposition were also accessed daily to ensure proper healing and recovery for 7 days post-surgery. In mortality cases, autopsy was performed to reveal the cause of death.

RESULTS
Overall, 42 mice underwent VSG (24 with antrum preservation, 14 without antrum preservation) for this study. Mice were euthanized for experiments at various times after surgery, and the stomach was harvested (Figure 2).
Because of the high mortality rate and low efficiency, VSG without antrum preservation was abandoned after 14 cases, and only the 24 cases of VSG with antrum preservation were included in the final analysis.

Body Weight Changes
Mean body weight at the time of operation was 28.9 grams. Postoperative body weight was lowest at postoperative week 1 (mean 24.1 grams; range 20-30 grams) and recovered after that, returning to its original weight and surpassing it thereafter. Although statistically not significant, body weights of VSG mice were always lower than non-operated control mice (Figure 3).

Mortality and Complications
There were no mortalities after VSG with antrum preservation, but 9 mice (64.3%) died during the early postoperative period after VSG without antrum preservation. The cause of death was anastomosis disruption secondary to gastric outlet obstruction in 8 cases ( Figure 4A) and pancreatic juice leakage in 1 case ( Figure 4B).

DISCUSSION
The mouse VSG technique described in this paper was developed to simplify and ease existing procedures without increasing postoperative mortality or morbidity. Two micro-clamps played a key role in the surgical technique of our VSG model. Originally, the clamp used in our model was designed for clamping small veins in human vascular surgery ( Figure 5). However, we  However, permanently placing clips on the stomach may restrict the natural recovery of gastric motility after VSG. Altered gastric motility after VSG has been known to be one of the key mechanisms of VSG, and many researchers focus on gastric motility changes after VSG in mice [6]. Therefore, the surgical model using permanent clipping does not appear to be suitable for the purpose of this study. Our model leaves behind no foreign material that may hinder the recovery or changes in gastric motility after surgery. Similar to our techniques, Hao et al. used a pair of custom-made stainless steel clamps for VSG [7]. However, there is no description on the clamp cost or how they are manufactured; therefore, this technique may not be available for most researchers.
The clamps used in this study are commercially readily available, cost-effective, and provide constant quality performance.
During the early phase of surgery, we tried both VSG with antrum preservation and without preservation. However, in cases of VSG without antrum preservation, there was such a high rate of complications that we abandoned the procedure after 14 cases; therefore, our result analysis only includes the antrum preservation cases. Most of the complications were related to gastric outlet obstruction, which was probably due to the transection and suturing being in close proximity to the pylorus. The mouse stomach is so small that it would be very difficult to obtain a true safety margin from the pylorus while achieving enough antrum resection. In humans, some surgeons prefer to remove the majority of the antrum for better outcomes, and they usually start the resection 2 to 3 cm from the pylorus [8]. However, in mouse VSG for study purposes, we recommend starting the resection right next to the pancreas gastric lobe so that the pylorus and pancreas remain intact after surgery.

CONCLUSIONS
Mouse VSG using two micro-clamps simplified existing VSG procedures by achieving field sterility and hemostasis with a single clamping.