The prevalence of diabetes and obesity is continuously increasing worldwide. The mechanisms between diabetes, obesity and their associated mortality and co-morbidities need to be further investigated. Here, we present a protocol for sleeve gastrectomy (SG) in animals as an uncomplicated preclinical model of bariatric surgery.
The number of people who are overweight and obese is continually increasing both in the adult and adolescent populations. This coincides with the increased universal phenomenon of type 2 diabetes (T2D) and other metabolic problems. Bariatric surgery, such as SG, is currently one of the most effective and commonly used long-term treatment for obesity and T2D, but the association between them is not completely explored yet. The mechanisms underlying the outcomes seen after bariatric surgery in humans can be investigated based on preclinical animal studies. The SG reduces body weight, glucose levels and many metabolic parameters, and is easy to perform with a low incidence of complications. The goal of this work is to provide a simple method and an uncomplicated preclinical model of bariatric surgery in animals for researchers.
The prevalence of obesity has increased nearly threefold worldwide since 1975. In 2016, more than 1.9 billion adults older than 18 years were overweight and over 650 million adults were obese. The prevalence of T2D in the adult population has also doubled from 4.7% to 8.5% with the number rising from 108 million to 422 million adults between 1980 and 2014 globally1. Most bariatric surgeries result in significant weight loss, a reduction of body fat, and improvement in glucose tolerance, insulin resistance and diabetes control2. In addition to weight loss and remission of T2D, bariatric surgery further produces additional health advantages such as hypertension control and a lower incidence of certain types of obesity related cancer development and progression3. Bariatric surgery also induces durable complete, partial remission of T2D, and decreases the risk of ten-year major coronary heart disease (CHD) and some degree of cerebrovascular risk. However, the underlying mechanisms are not completely understood4.
There are several procedures for bariatric/metabolic surgery. Both restrictive and gastrointestinal bypass surgeries provide positive effects on metabolism5. Of the bariatric surgical models, SG and modified Roux-en-Y gastric bypass have higher success and lower mortality rates and demonstrate reliable restrictive and gastrointestinal bypass surgery models in mice6. Although the gastrointestinal bypass surgery provides more weight loss and a significant improvement of glucose tolerance and liver steatosis than the restrictive procedure in the long term, the SG still produces good control of body weight and glucose levels and is easy to perform with a low incidence of complications6. The proportion of SG increased from 30% to 54% and Roux-en-Y gastric bypass surgery decreased from 52% to 32% from 2008 to 20147. Currently, laparoscopic SG is the most commonly performed bariatric procedure at the national level within academic centers in the United States8. Although there have been many published reports regarding the pathophysiological processes between bariatric surgery, diabetes and obesity, we need more animal experiments to further explore unknown mechanisms.
This protocol aims to produce an animal method to investigate the mechanisms underlying the outcomes seen after bariatric surgery in humans. The current translational study may provide insights on the mechanism of obesity and T2D treatment from the SG.
All procedures for animal use were approved by the National Yang-Ming University Institutional Animal Care and Use Committee and complied with the “Guide for the Care and Use of Laboratory Animals. 8th edition, 2011”. The anesthetic procedure was performed according to the guideline for the pain control, anesthesia, pre-operative, and postoperative care for the experimental animals from National Yang Ming University, ICAUC-016, 2015, 1st edition.
1. Animal preparation
2. General preoperative preparation
3. Sleeve gastrectomy and sham procedures
4. Stomach isolation and clip
NOTE: The SG for mice was performed using the new clip applier technique as previously published10.
5. Sham procedure of SG
6. General postoperative care
7. Metabolic parameter assessment
Results of the operation are shown in Figure 3 and Figure 4. The survival rate of the study was 90%. One mouse died in the sham group because of weakness and another mouse died in the SG group on the third day after operation for unknown reasons. Results obtained on weight loss (Figure 3A) after operation (at week 3) on HFD fed mice were quite similar to those observed in humans, with a final body weight loss of about 15-20%13. The study also demonstrated that the cholesterol level (Figure 3B) decreased significantly after SG surgery in the HFD fed mice.
Better insulin sensitivity and glucose tolerance were observed soon after the surgery by an intraperitoneal glucose tolerance test (IPGTT). In the current study, we demonstrated that the HFD-induced obesity evoked insulin resistance and glucose intolerance in wild type C57BL/6 mice and can be corrected by SG. After SG, the insulin resistance, glucose, and lipid levels all improved in this study.
Figure 1: Surgical procedures of SG.
(A) The 5 mm surgical clip used in this experimental model. (B) Check the depth of anesthesia with pinch stimulation. (C,D) Apply the clip to the half of medial side of the stomach midline from gastroesophageal junction inferiorly and lower pole superiorly. (E) Sterilize and suture the cut edge of the stomach to ensure no leakage. (F–H) Hydrate the mice with warm saline, return the stomach and intestine back to the proper site in the abdominal cavity. Close the abdominal cavity carefully. Recover from the anesthetizing status in the induction chamber and return to the cage when the condition was fully recovered and stabilized. Please click here to view a larger version of this figure.
Figure 2: Sites of surgical clip usage to the stomach. Please click here to view a larger version of this figure.
Figure 3: The effect of SG on (A) weekly body weight changes and (B) cholesterol level.
Data are represented as mean ± SD. *P < 0.05; **P < 0.001. Please click here to view a larger version of this figure.
Figure 4: Effect of SG on (A) IPGTT (B) Hba1c and (C) HOMA-IR at the end of the study.
(n = 5 VSG; n = 7 sham); Data are represented as mean ± SD. *P < 0.05; **P < 0.001. Please click here to view a larger version of this figure.
Most of the prospective cohort studies have confirmed that increasing the body mass index is related to increasing the mortality. Obesity is associated with a higher risk of diabetes, cardiovascular diseases (CVD), and death in both sexes. The specific pathologic processes have established a link between diabetes, obesity, and mortality14. The systematic reviews indicate the benefits of bariatric surgery in reducing risk factors for CVD with evidence for ventricular hypertrophy regression and improved diastolic function as well15. Bariatric/metabolic surgery can improve traditional CVD risk factors in obese diabetic patients, and the 10-year CHD and fatal CHD risk have shown to be reduced by up to 50% after various bariatric/metabolic surgical procedures in obese patients with T2D4.
Bariatric surgery is the most effective long-term therapeutic strategy for obesity. The significant improvements of metabolic parameters from bariatric surgery such as lipid profile, inflammatory biomarkers, blood pressure, and heart failure are also noted especially in the unexpected type 2 diabetic control. SG has reduced and modified adipose tissue inflammation and development and has possibly reduced the atherosclerosis risk.
Changes of gastro-intestinal hormones might play a role in long-term weight control16,17. The SG has also reduced caloric intake and normalized the glycemic control18. The gastric bypass surgery related glycemic tolerance effects are related to the foregut hormonal effects evoked by bypassing the proximal bowel from the current theories19. However, the SG has demonstrated the comparable antidiabetic effect, suggesting further non-bypassing related unknown mechanisms even beyond the effect of glucagon-like peptide 1 receptor16,20. Lopez et al. have shown that SG promotes weight loss, improves the cholesterol and glucose profile, leptin insensitivity, and decreases insulin resistance, which is independent of the ghrelin level changes in obese Zucker rats21. All these above issues need further animal studies to clarify the connection with diabetes, obesity and diseases. SG is a bariatric procedure that does not involve intestinal bypass, which has become increasingly popular in recent decades in the US and globally due to low long term complications22.
The bariatric surgical procedure is technically difficult and time consuming with high surgical mortality and long-term complication and is the main critical step for small animal experiments. The SG is currently the most used surgery for weight reduction in the US because it is a simple procedure with high effectiveness, low co-morbidity, and low mortality. This protocol has similar advantages mimicking the weight reduction and metabolic surgeries practiced routinely in humans. In general, surgical techniques are quite difficult in mice because of the small body size, and a dissecting microscope is frequently needed with high surgical mortality.
Schlager et al. have developed a mouse model of SG with a surgical clip application instead of a hand sewn closure for the stomach. This animal model has demonstrated similar effects of body weight reduction and glucose control, but has failed to reveal significant pancreatic islet cell proliferation10. This effect on the metabolism from this mice model is similar to the human bariatric/metabolic surgery with a very low operative mortality.
This current study demonstrates constant weight reduction after the SG in the HFD fed mice with improved glycated hemoglobin, IPGTT, HOMA-IR (homeostatic model assessment for insulin resistance), and cholesterol level. This surgical procedure is safe and easy to perform in mice.
The surgical survival rate of this protocol in our laboratory is around 90% after a learning period, and the procedure takes less than 15 minutes. This protocol may provide significant value in future small rodent studies with surgical feasibility, safety, reproducibility, and clinical relevance. This method may also be used to highlight physiological changes observed after bariatric surgery in humans. The difficulty of SG surgery plays an important factor in the success of animal studies. This protocol is easy to perform, and imitates the SG as commonly practiced in other animal and human studies.
This animal model can investigate the clinical relevance of diabetes and obesity using a brief surgical procedure with sleeve gastrectomy. These procedures can shorten the operation time and improve the surgical survival rate. The main advantage of this study is to mimic the clinical outcomes of bariatric surgery in human patients using a simple surgical model.
The authors have nothing to disclose.
We thank Miss Isabelle Lu for the English narration.
0.9% normal saline | China Chemical & Pharmaceutical Co., Ltd. | ||
5-0 monofilament suture | Shineteh Inc. Taiwan | Ethicon | |
Alm | Shineteh Inc. Taiwan | ST-A072PK | UNIK |
Animal Anesthesia | Step Technology | 0712VAP11076 | Matrx |
Barraqer-Troutman forceps | Shineteh Inc. Taiwan | ST-N309(H-4410) | UNIK |
cefazolin | Taiwan veteran pharmacy company | ||
Chow diet | Research Diets | ||
Glucose strips and glucometer | BeneCheck | BKM13-1 | BeneCheck |
HbA1c kit | Level | OKEH00661 | |
Hematology system | Fuji | FUJI DRI-CHEM 4000i | Europe |
High Fat diet | Research Diets | 1810724 | LabDiet |
Iris Forceps | Shineteh Inc. Taiwan | ST-1210 | UNIK |
Iris Scissors | Shineteh Inc. Taiwan | ST-S011 | UNIK |
Isoflurane | Panion & BF biotech INC | 8547 | Panion & BF |
Ketoprofen | Sigma | 22071154 | Sigma-Aldrich |
Stereo microscope | MicroTech | SZ-5T | MicroTech |
Surgical clip (M) | Echicon Inc., Somerville, NJ | Size M, 5mm | |
Vidisic gel | Dr. Gerhard Mann Chem.-pharm. Fabrik GmbH |