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

International Microsurgery Journal. 2020;4(1):5
DOI: 10.24983/scitemed.imj.2020.00137
Article Type: How I Do It

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.

Keywords

  • Micro-clamps; mouse; technique; vertical sleeve gastrectomy

Video 

Introduction

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.

Surgical Techniques

All experimental protocols have been approved by the University of Nevada, Reno Institutional Animal Care and Committee (protocol #00727).

Preoperative Preparation
All laboratory mice were purchased from Jackson Laboratories (Bar Harbor, ME, United States). Male C57BL mice, aged 8 weeks, and with a body weight 28 to 30 grams were used for VSG. Mice were fed ad libitum and were placed on a liquid-only diet (chocolate-flavor Boost®, Nestle, Vevey, Switzerland) 3 days before surgery. During the liquid diet period, cages were embedded with paper cellulose particles (Alpha dri, Shepherd Specialty Papers, Milford, NJ, United States) to minimize pica.

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 (O2) 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 O2 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 https://doi.org/10.24983/scitemed.imj.2020.00137 for details of the procedure). 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.

Figure 1. (A) Stomach was exteriorized after omentum clearance. Note the distended stomach after 3 days of liquid diet. (B) Two micro-clamps were applied in both directions to delineate the line of transection, and (C) the greater curvature side was removed. Note the gastrotomy for stomach evacuation.


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).
 

Figure 2. The vertical sleeve gastrectomy with antrum preservation specimen after 4 months of surgery (d, duodenum; p, pylorus; s, stomach; e, esophagus).

 

Figure 3. Body weight changes after surgery, measured on a daily basis for 1 week and then at 2 and 4 weeks. Note that the body weight of mice with vertical sleeve gastrectomy was always lower than that of control mice (wild-type, WT).

 

Figure 4. (A) Gastric outlet obstruction was a typical complication after vertical sleeve gastrectomy without antrum preservation. Note the severely distended stomach. This complication was occasionally complicated by anastomosis perforation. (B) This mouse died of pancreatic juice leakage after vertical sleeve gastrectomy without antrum preservation. Note the self-digestion by pancreatic juice.

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 found that this clamp was also useful for experimental mouse surgery. The clamp conveniently served as both a non-crushing tissue clamp and a vascular clamp for anastomosis and hemostasis in mouse VSG.
 

Figure 5. The plastic, disposable micro-clamp was designed for vascular surgery in humans.


Garibay et al. described a technique of placing a continuous suture along the intended line of transection to prevent spillage of gastric contents before transection [4]. Also, they had ligated prominent branches of the gastric artery and vein before transection. However, placing such sutures is a time-consuming procedure in itself, and proceeding with surgical manipulation without completely clearing the gastric contents may cause potentially fatal abdominal sepsis after surgery. In our procedure, we could prevent intraoperative spillage and bleeding by maintaining clamps on the cut edge of the stomach throughout the course of the surgery. Schlager et al. showed a rather simple technique by applying 2 permanent titanium clips, instead of suturing, to close the cut edge of the stomach [5]. This technique requires only a few sutures to close the gap between the 2 clips, and the operation time would be shorter than ours. 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.

References

  1. Varela JE, Nguyen NT. Laparoscopic sleeve gastrectomy leads the U.S. Utilization of bariatric surgery at academic medical centers. Surg Obes Relat Dis 2015;11(5):987-990. [View Article]
  2. Bozadjieva N, Heppner KM, Seeley RJ. Targeting FXR and FGF19 to treat metabolic diseases-Lessons learned from bariatric surgery. Diabetes 2018;67(9):1720-1728. [View Article]
  3. Stevenson M, Lee J, Lau RG, Brathwaite CEM, Ragolia L. Surgical mouse models of vertical sleeve gastrectomy and Roux-en Y gastric bypass: A review. Obes Surg 2019;29(12):4084-4094. [View Article]
  4. Garibay D, Cummings BP. A murine model of vertical sleeve gastrectomy. J Vis Exp 2017(130):e56534. [View Article]
  5. Schlager A, Khalaileh A, Mintz Y, et al. A mouse model for sleeve gastrectomy: Applications for diabetes research. Microsurgery 2011;31(1):66-71. [View Article]
  6. Chambers AP, Smith EP, Begg DP, et al. Regulation of gastric emptying rate and its role in nutrient-induced GLP-1 secretion in rats after vertical sleeve gastrectomy. Am J Physiol Endocrinol Metab 2014;306(4):E424-432. [View Article]
  7. Hao Z, Townsend RL, Mumphrey MB, Morrison CD, Munzberg H, Berthoud HR. RYGB produces more sustained body weight loss and improvement of glycemic control compared with VSG in the diet-induced obese mouse model. Obes Surg 2017;27(9):2424-2433. [View Article]
  8. Avlanmis O, Isil RG, Burcu B. Effect of resection distance from pylorus on weight loss outcomes in laparoscopic sleeve gastrectomy. Obes Surg 2019;29(9):2731-2738. [View Article]

Editorial Information

Publication History

Received date: May 10, 2020
Accepted date: September 07, 2020
Published date: September 23, 2020

Funding

This study was supported by a 2017 research grant from Kangwon National University (grant no. 520170434). 

Conflict of Interest

The authors report no financial or other conflict of interest relevant to this article, which is the intellectual property of the authors.

Copyright

© 2020 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY).

Department of Surgery, Kangwon National University School of Medicine, Chuncheon, South Korea
Department of Health Management and Policy, Kangwon National University School of Medicine, Chuncheon, South Korea
Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, Nevada, USA
Department of Surgery, Kangwon National University School of Medicine, Chuncheon, South Korea

Address: 1 Gangwondaehak-gil, Hyoja-dong, Chuncheon-si, Gangwon-do, South Korea
Figure 1.JPG
Figure 1. (A) Stomach was exteriorized after omentum clearance. Note the distended stomach after 3 days of liquid diet. (B) Two micro-clamps were applied in both directions to delineate the line of transection, and (C) the greater curvature side was removed. Note the gastrotomy for stomach evacuation.
Figure 2.jpg
Figure 2. The vertical sleeve gastrectomy with antrum preservation specimen after 4 months of surgery (d, duodenum; p, pylorus; s, stomach; e, esophagus)
Figure 3.jpg
Figure 3. Body weight changes after surgery, measured on a daily basis for 1 week and then at 2 and 4 weeks. Note that the body weight of mice with vertical sleeve gastrectomy was always lower than that of control mice (wild-type, WT).
Figure 4.JPG
Figure 4. (A) Gastric outlet obstruction was a typical complication after vertical sleeve gastrectomy without antrum preservation. Note the severely distended stomach. This complication was occasionally complicated by anastomosis perforation. (B) This mouse died of pancreatic juice leakage after vertical sleeve gastrectomy without antrum preservation. Note the self-digestion by pancreatic juice.
Figure 5.jpg
Figure 5. The plastic, disposable micro-clamp was designed for vascular surgery in humans.

Reviewer 1 Comments

  1. This is a nicely written article, but some major issues need to be addressed. First, the aim of the study is vague, and the authors must specify it in the Introduction section.
    ResponseThe aim of this study is to introduce more simplified technique to perform VSG, and we modified the last sentence of Introduction, and as follow. To overcome such demerits and to simplify the existing techniques, we describe herein a modified technique of mouse VSG using 2 disposable micro-clamps.
  2. The definition of the angle of His may be added in the article. For instance, the angle of His is the acute angle created between the first part of the stomach at the entrance to the stomach and the esophagus.
    ResponseIt would be inappropriate to mention the definition of angle of His in this article, so we decided not to comment on the angle of His.
  3. There were 24 cases of VSG with antrum preservation included in the final analysis. Were these 24 cases consecutive or were there any cases that were excluded between these cases?
    ResponseAll cases were consecutive, and we modified the Results as follow. Overall, 42 mice underwent VSG (24 consecutive cases with antrum preservation, 14 consecutive cases without antrum preservation) for this study.
  4. How many wild-type mice were included in the study?
    ResponseAll of the mice used in this study were wild type, and we modified the sentence as follow. Wild-type, male C57BL mice, aged 8 weeks, and with a body weight 28 to 30 g were used for VSG.
  5. In Figure 3, is the difference in body weight between the study and the controlled groups significant?
    ResponseThe difference in body weight was not statistically significant, and we chenged the sentence in Results, and as follow. Although statistically not significant, body weights of VSG mice were always lower than non-operated control mice (Figure 3).
     

Reviewer 2 Comments

  1. The authors stated that most researchers studying such mechanisms use a mouse model of VSG, and there have been various surgical techniques to perform VSG in mice safely and effectively, each with their own advantages and disadvantages. It is better to clarify the so-called “advantages and disadvantages”. In fact, the authors should provide a brief review of the literature addressing the limitations and/or flaws in previous studies; the unresolved gaps in the research; and/or contradictory outcomes between studies. In other word, the research question must be clearly defined for a more readable and understandable purpose.
    ResponseDisadvantages of the existing techniques were mentioned in detain in the 2nd paragraph of Discussion. However, to clearly show the purpose of this study, we briefly mentioned the demerits of existing procedure and stated the purpose of this article with clarity and as follow. However, some techniques required tedious, time-consuming 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.
     

Reviewer 3 Comments

  1. The results of the study should provide an answer to the study hypotheses or research issues outlined in the Introduction. The authors reported a modified technique with 2 disposable micro-clamps to simplify the vertical sleeve gastrectomy to reduce operation time and enhance technical efficiency. Nevertheless, I can not see the data to explain how this modified technique could save surgical time and improve its technical efficiency. The authors should clarify this major issue and describe it in the Abstract section and Results section.
    ResponseTo delineate the line of transection and to prevent spillage, most of the existing VSG techniques uses continuous suture along the gastric wall before gastrectomy (ref. #3,4). The main issue of this study is to show the feasibility of applying 2 micro-clamps instead of such a tedious, time-consuming suture procedure. Although this is not a comaprative study comparing previous techniques and our technique, using our modified technique, we observed satisfactory weight loss without mortality (in antrum preservation cases) after VSG. We speculate that this is a kind of qualitative issue, not a quantitative issue. Also, we deleted the result on the operation time in Abstract and Results and the related sentence in Abstract was changed and as follow. Mean body weight at postoperative week 1 was 24.1 g, which is equivalent to a 16.1% loss of original body weight, and there was no mortality after antrum prevation VSG.
     

Reviewer 4 Comments

  1. I thoroughly read the manuscript. I suggest that the authors should revise it given some issues to be clarified. I hope that the authors could revise the manuscript so that it cab be more complete, satisfactory to be able to be published. First, what is main information you want to publish? With the title of the manuscript, as well as what presented and emphasized in sections of “Introduction” and “Discussion”, it looks that you want to introduce to readers your modified experimental technique of vertical sleeve gastrectomy on mouse, with an outstanding detail being the use of 2 disposable micro-clamps. However, the content of the manuscript didn't totally focus onto this target.
    ResponseIntroduction section was revised to clearly show the purpose of this study, and as follow. 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.
     
  2. The research objective was not clear in the Introduction section. Hence, the contents stated in the “Conclusions” section may not be congruent.
    ResponseTo focus on the main objective of this study, we had deleted comments on the complications after VSG at Conclusion section.
     
  3. A small heterogeneity in word usage: while in the title is "Modified techniques ...", you used "a modified technique..." in the content of the manuscript.
    ResponseTitle was revised to improve consistency in word usage, and as follow. A Modified Technique of Mouse Vertical Sleeve Gastrectomy Using Two Disposable Micro-Clamps.
     

Reviewer 5 Comments

  1. Authors must specify the aim of the study. For example, in the Abstract section, the aim of the study was to reduce operation time and enhance technical efficiency, and the key aspects of the modification were to use the micro-clamps as non-crushing tissue clamps for anastomosis as well as vascular clamps for hemostasis. In the Introduction section, the purpose of the study was to describe a modified technique of mouse VSG using 2 disposable micro-clamps, which is simple to perform, readily reproducible with cost-effectiveness, and faster in operation time. However, in the Results section, the operation time was the only variable of interest, the measurement scale for defining the technical efficiency was lacking. Also, were the results of operation time shorter in comparison to what controlled objects?
    Response We had deleted redundant comments on the operation time and technical efficiency, which could not be quantitatively demonstrated in this article, and the related sentences in Abstract were revised and as follow. 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.

    We had modified the last paragraph of Introduction to cleraly state the purpose of this study, and as follow. 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.

    We totally agree that the improvement in the technically efficienty could not be demonstrated quantitatively within this study, and we had removed comments on technical efficinecy throughout the manuscript. We also agree that the operation time has no counterpart to compare with. Therefore, we had deleted the results on operation time in Result section, and comments on the operation time were removed throughout the manuscript.
     

Editorial Comments

  1. The authors should re-submit the figures without the labels (A), (B), and (C).
    ResponseWe had removed the labels.
     
  2. The resolution of the video is low. It would be better to re-upload a high-quality video.
    ResponseWe are sorry that we do not to have a better resolution video.