This research was supported by NIH/NCI R21CA195002-01A1, The President&#39;s Council of Cornell Women and the SUNY Graduate Diversity Fellowship. Dr. Cummings&#39; laboratory also received funding during the project period from the Cornell Comparative Cancer Biology Training Program and Eli Lilly and Company.

All experimental protocols have been approved by the Cornell University Institutional Animal Care and Use Committee.
1. Pre-surgical Preparation
NOTE: Study mice are typically on a C57BL diet-induced obese background to make studies translationally relevant to human obesity and insulin resistance. Male and female mice may be studied as described in the subsequent steps.
<ol>
	<li>At an age of 2 months, place mice on a 45% or 60% high fat diet for 2 months (see Table of Materials). 
	NOTE: Mice are fed a high fat diet to create an obese and insulin resistant phenotype. Shorter or longer periods of high fat diet feeding may be used depending on the goals of the study.</li>
	<li>Continue to feed mice ad libitum until surgery. If baseline measurements are needed, after 7 weeks of high fat diet feeding, fast mice for 6 h or overnight and take a baseline blood sample for measurement of metabolites of interest (e.g., glucose).</li>
	<li>Continue mice on high fat diet and allow mice one week to recover after blood collection prior to sham surgery or VSG. Place mice on a liquid diet (see Table of Materials) for 4 days before surgery. 
	NOTE: Mice are maintained on a liquid diet before and after surgery to clear the digestive tract of particulate matter that may impair the healing response.</li>
	<li>Sterilize the following items prior to surgery: foil, gauze, cotton tipped applicators (CTAs) and surgical instruments (hemostat, operating scissor, iris scissor, dumont forcep, serrated Brown-Adson forcep, gavage needle, microneedle driver, spring scissor).</li>
</ol>
2. Vertical Sleeve Gastrectomy and Sham Procedures
<ol>
	<li>On the day of surgery weigh mice to obtain a baseline weight measurement.</li>
	<li>Equip the surgical room with an anesthesia system, heated water bath, and heating pad. Set the water bath to 37 &#176;C and use it to warm 0.9% saline solution for irrigation. Clean the surgical field with 70% ethanol and use sterile technique to open autoclaved surgery pack and tools. 
	NOTE: The heating pad is used to keep the animal warm during surgery and recovery from anesthesia.</li>
	<li>Using an induction chamber, anesthetize the mouse with 5% isoflurane and O2 flow rate of 1 L/min. Confirm that the mouse is in the appropriate plane of anesthesia by performing a toe pinch. Maintain the mouse with 1-3% isoflurane and an O2 flow rate of 0.6 L/min once a consistent plane of anesthesia has been achieved.</li>
	<li>Once the mouse is anesthetized, place eye ointment (see Table of Materials) on its eyes to keep them moist during surgery. At this time also administer an analgesic, such as meloxicam (2 mg/kg subcutaneously). Clip the hair from the umbilicus to the axilla holding the skin down gently to provide tension.
	<ol>
		<li>As the mouse&#39;s skin is thin and delicate, take care to not cause skin lesions when clipping the hair. Clean the skin with povidone-iodine and alcohol.</li>
		<li>Don sterile gloves and prepare the sterile surgical field and instruments. Perform the rest of the procedure using sterile technique. Prepare the sterile surgical field by placing sterile surgical drapes on either side of the mouse. Use autoclaved tin foil to create a surgical drape for the mouse.</li>
		<li>Cut a small hole in the tin foil to allow access to the mouse&#39;s abdomen.</li>
	</ol>
	</li>
	<li>Make an incision of the skin from the mid-abdomen (umbilicus) to the level of the xiphoid cartilage using an iris scissor. Identify the linea alba and use iris scissors to cut through the body wall along the linea alba. Use CTAs to gently elevate the stomach out of the abdomen and then bluntly dissect the greater omentum off of the greater curvature of the stomach.</li>
	<li>Ligate the short gastric artery that runs between the fundus of the stomach and the spleen by placing two ligatures using 7-0 monofilament absorbable suture. Use spring scissors to cut between the two ligatures. Once the artery is transected, fully exteriorize the stomach from the abdominal cavity. Place gauze under the stomach and wet with saline to keep the tissue moist.</li>
	<li>Sham Procedure
	<ol>
		<li>For the sham procedure, place a loose simple continuous pattern of suture using 6-0 monofilament absorbable suture with a taper needle. Begin 2 mm to the right (surgeon&#39;s right) of the esophagus. Place the suture line along the ventral gastric wall and then continue along the dorsal gastric wall. 
		NOTE: For all gastric manipulations, use only monofilament absorbable suture to decrease the risk of infection. Furthermore, use only suture with a taper needle for all gastric manipulations, as use of a cutting needle increases the risk of suture &#34;pull-through&#34; leading to gastric tissue damage and wound dehiscence.</li>
		<li>Pass the needle completely through one gastric wall while placing the suture. Make sure the suture is lying flat, but not restricting the stomach. Gently tie the suture off using 3-4 throws and skip to step 2.9.</li>
		<li>For the VSG procedure skip step 2.7 and proceed with step 2.8.</li>
	</ol>
	</li>
	<li>VSG procedure
	<ol>
		<li>For the VSG procedure, ligate the prominent branches of the gastric artery and vein with 7-0 monofilament absorbable suture with a taper needle using 3 throws for each knot. Place the ligatures just below (i.e., towards the lesser curvature of the stomach) the intended line of transection. 
		NOTE: The intended line of transection starts at ~2 mm above (meaning towards the greater curvature of the stomach) the cardiac notch of the stomach and at least 2 mm below the margo pilcatus and extends to the proximal end of the right lobe of the pancreas. Typically, there are 4 vessels per gastric wall that need to be ligated; however, this may vary between mice.</li>
		<li>To prevent spillage of gastric contents during the gastrectomy, place a simple continuous line of suture passing through both gastric walls just below the intended line of transection using 6-0 monofilament absorbable suture with a taper needle. Begin the suture line to the surgeon&#39;s right of the esophagus and below the margo pilcatus and end just above the pancreas.</li>
		<li>Place thin-tipped hemostats above the suture line and use spring scissors to cut between the suture line and hemostats. Remove the transected gastric tissue from the sterile surgical field. Use CTAs to clean blood and digesta off of the stomach.</li>
		<li>Reinforce apposition of the gastric walls using 6-0 monofilament absorbable suture with a taper needle using a simple discontinuous pattern. 
		NOTE: A simple discontinuous suture pattern is recommended since this provides more secure closure than the simple continuous suture pattern.</li>
		<li>Flush the gastric remnant with saline throughout the procedure in order to keep the tissue moist and clean. Use a 20G gavage needle attached to a 20-mL syringe to perform gastric lavage with saline. Use this size to ensure adequate pressure is provided without risking gastric tissue damage from excessive pressure or accidental gastric tissue damage from use of a regular pointed needle.</li>
		<li>Use a minimum of 20 knots to securely close the stomach; pay attention to closure along the esophageal side, as this side is more difficult to access and therefore is often a site of dehiscence.</li>
		<li>Ensure that there are no leaks by gently pressing on the stomach with CTAs. If leaks are identified, place additional simple discontinuous knots on the areas of leakage and then leak test again. Leak test until no leakage is detected.</li>
		<li>Do a final thorough lavage of the stomach using at least 60 mL of saline to ensure that no infectious particulate matter has been left behind.</li>
	</ol>
	</li>
	<li>Place the stomach back into the abdominal cavity under the liver using a CTA. Place CTAs along the dorsal aspect of the abdominal cavity to absorb all excess fluid. Using a blunt 18G needle, inject lactated Ringer&#39;s solution (LRS) with or without antibiotics (0.5 mL LRS +/- 20 mg/kg Enrofloxacin) directly into the abdominal cavity, just prior to closure. 
	NOTE: This replaces fluid loss experienced during surgery and provides a method for direct application of antibiotics to the surgical site to assist with recovery.</li>
	<li>Close the abdominal muscle layer using 6-0 monofilament absorbable suture with a taper needle in a simple discontinuous pattern. Then close the skin layer with 6-0 monofilament absorbable suture in a simple continuous pattern.</li>
	<li>Place tissue adhesive on the skin and fold the skin over the suture line to bury the suture so that the mouse cannot disrupt the wound closure post-operatively.</li>
	<li>Turn the isoflurane off and let the mouse recover on the heating pad for 10-15 minbefore returning it to its home cage. Do not leave the animal unattended until it has regained consciousness and is able to move.</li>
	<li>Keep animals housed singly until fully recovered from surgery. 
	NOTE: If accurate food intake measurements are to be obtained throughout study, mice should continue to be singly housed throughout study. There is a risk of foreign body obstruction from eating home cage bedding. Animals should be housed in cages free of bedding except for nesting material to provide enrichment (see Table of Materials).</li>
</ol>
3. Post-operative Mouse Care and Measurements
<ol>
	<li>Return the mouse to its home cage and place the cage on a heating pad. Maintain mice on a liquid diet for at least 7 days after surgery. Administer an analgesic, such as meloxicam (2 mg/kg subcutaneously), for 2 days after surgery. 
	NOTE: An antibiotic, such as enrofloxacin (20 mg/kg), may be given for up to 7 days after surgery. Antibiotics are given to minimize risk of infection from the surgical procedure and may or may not be required depending on the level of sterility maintained throughout the procedure. However, if antibiotics are used, they must be administered to all mice in the same study to maintain consistency.</li>
	<li>During the post-operative period assess body weight, food intake, defecation, activity level, and disposition at least daily to ensure proper healing and recovery. Conduct post-operative monitoring for 14 days after surgery. 
	NOTE: Surgical failure is typically seen either immediately after surgery or within three days post- operatively. Animals that are not eating or drinking on the second day after surgery must be closely monitored for surgical complications.</li>
	<li>After successful completion of the post-operative period, measure body weight and food intake regularly throughout study to ensure maintenance of the appropriate phenotype.</li>
	<li>Perform an oral glucose tolerance test (OGTT) to assess glucose tolerance, glucose-stimulated insulin secretion, and gut hormone secretion.
	<ol>
		<li>Perform the OGTT in mice after fasting them for 6 h and then gavage mice with 50% dextrose solution at a 1 g/kg dose.</li>
		<li>Collect tail blood samples at 0, 2.5, 5, 15, 30, 60, and 120 minutes after the gavage. Measure blood glucose with a glucometer at these time points. Collect serum samples at each time point for measurement of various hormones, such as insulin and GLP-1.</li>
		<li>Fast mice for 6 h prior to euthanasia. Take a final blood sample and glucose measurement. Compare the final glucose measurement to the baseline measurement. Euthanize mice with an intraperitoneal injection of pentobarbital at a 200 mg/kg dose.</li>
		<li>Use the baseline and final serum samples to measure additional analytes and compare pre- and post-operative surgical values.</li>
	</ol>
	</li>
</ol>

<ol><li>Buchwald, H., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/15479938">Bariatric surgery: a systematic review and meta-analysis.</a> JAMA. 292 (14), 1724-1737 (2004).</li>
<li>Wadden, T. A., Butryn, M. L., Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+of+lifestyle+modification+for+long-term+weight+control%5BTitle%5D)+AND+%22Obes+Res%22%5BJournal%5D)">Efficacy of lifestyle modification for long-term weight control.</a> Obes Res. 12, Suppl . 151s-162s (2004).</li>
<li>Yanovski, S. Z., Yanovski, J. A. <a target="_blank" href="http://www.nejm.org/doi/full/10.1056/NEJMra012586">Obesity.</a> N Engl J Med. 346 (8), 591-602 (2002).</li>
<li>Abbatini, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+effects+of+laparoscopic+sleeve+gastrectomy%2C+gastric+bypass%2C+and+adjustable+gastric+banding+on+type+2+diabetes%5BTitle%5D)+AND+%22Surg+Endosc%22%5BJournal%5D)">Long-term effects of laparoscopic sleeve gastrectomy, gastric bypass, and adjustable gastric banding on type 2 diabetes.</a> Surg Endosc. 24 (5), 1005-1010 (2010).</li>
<li>Sjostrom, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+bariatric+surgery+on+mortality+in+Swedish+obese+subjects%5BTitle%5D)+AND+%22N+Engl+J+Med%22%5BJournal%5D)">Effects of bariatric surgery on mortality in Swedish obese subjects.</a> N Engl J Med. 357 (8), 741-752 (2007).</li>
<li>Adams, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+incidence+and+mortality+after+gastric+bypass+surgery%5BTitle%5D)+AND+%22Obesity+%28Silver+Spring%29%22%5BJournal%5D)">Cancer incidence and mortality after gastric bypass surgery.</a> Obesity (Silver Spring). 17 (4), 796-802 (2009).</li>
<li>Vest, A. R., Heneghan, H. M., Schauer, P. R., Young, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+management+of+obesity+and+the+relationship+to+cardiovascular+disease%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Surgical management of obesity and the relationship to cardiovascular disease.</a> Circulation. 127 (8), 945-959 (2013).</li>
<li>Ahmed, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laparoscopic+Roux-en-Y+gastric+bypass+and+its+early+effect+on+blood+pressure%5BTitle%5D)+AND+%22Obes+Surg%22%5BJournal%5D)">Laparoscopic Roux-en-Y gastric bypass and its early effect on blood pressure.</a> Obes Surg. 19 (7), 845-849 (2009).</li>
<li>Pories, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Who+would+have+thought+it%3F+An+operation+proves+to+be+the+most+effective+therapy+for+adult-onset+diabetes+mellitus%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Who would have thought it? An operation proves to be the most effective therapy for adult-onset diabetes mellitus.</a> Ann Surg. 222 (3), 339-350 (1995).</li>
<li>Garibay, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-Cell+Glucagon-Like+Peptide-1+Receptor+Contributes+to+Improved+Glucose+Tolerance+After+Vertical+Sleeve+Gastrectomy%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">beta-Cell Glucagon-Like Peptide-1 Receptor Contributes to Improved Glucose Tolerance After Vertical Sleeve Gastrectomy.</a> Endocrinology. 157 (9), 3405-3409 (2016).</li>
<li>McGavigan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGR5+contributes+to+glucoregulatory+improvements+after+vertical+sleeve+gastrectomy+in+mice%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">TGR5 contributes to glucoregulatory improvements after vertical sleeve gastrectomy in mice.</a> Gut. 66 (2), 226-234 (2017).</li>
<li>McGavigan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vertical+sleeve+gastrectomy+reduces+blood+pressure+and+hypothalamic+endoplasmic+reticulum+stress+in+mice%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">Vertical sleeve gastrectomy reduces blood pressure and hypothalamic endoplasmic reticulum stress in mice.</a> Dis Model Mech. 10 (3), 235-243 (2017).</li>
<li>Esteban Varela, J., Nguyen, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laparoscopic+sleeve+gastrectomy+leads+the+U.S.+utilization+of+bariatric+surgery+at+academic+medical+centers%5BTitle%5D)+AND+%22Surg+Obes+Relat+Dis%22%5BJournal%5D)">Laparoscopic sleeve gastrectomy leads the U.S. utilization of bariatric surgery at academic medical centers.</a> Surg Obes Relat Dis. 11 (5), 987-990 (2015).</li>
<li>Laferrere, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+weight+loss+by+gastric+bypass+surgery+versus+hypocaloric+diet+on+glucose+and+incretin+levels+in+patients+with+type+2+diabetes%5BTitle%5D)+AND+%22J+Clin+Endocrinol+Metab%22%5BJournal%5D)">Effect of weight loss by gastric bypass surgery versus hypocaloric diet on glucose and incretin levels in patients with type 2 diabetes.</a> J Clin Endocrinol Metab. 93 (7), 2479-2485 (2008).</li>
<li>Adams, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Health+benefits+of+gastric+bypass+surgery+after+6+years%5BTitle%5D)+AND+%22Jama%22%5BJournal%5D)">Health benefits of gastric bypass surgery after 6 years.</a> Jama. 308 (11), 1122-1131 (2012).</li>
<li>McGavigan, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGR5+contributes+to+glucoregulatory+improvements+after+vertical+sleeve+gastrectomy+in+mice%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">TGR5 contributes to glucoregulatory improvements after vertical sleeve gastrectomy in mice.</a> Gut. , (2015).</li>
<li>Peterli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improvement+in+glucose+metabolism+after+bariatric+surgery%3A+comparison+of+laparoscopic+Roux-en-Y+gastric+bypass+and+laparoscopic+sleeve+gastrectomy%3A+a+prospective+randomized+trial%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Improvement in glucose metabolism after bariatric surgery: comparison of laparoscopic Roux-en-Y gastric bypass and laparoscopic sleeve gastrectomy: a prospective randomized trial.</a> Ann Surg. 250 (2), 234-241 (2009).</li>
<li>Ryan, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FXR+is+a+molecular+target+for+the+effects+of+vertical+sleeve+gastrectomy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">FXR is a molecular target for the effects of vertical sleeve gastrectomy.</a> Nature. 509 (7499), 183-188 (2014).</li>
<li>Wilson-Perez, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vertical+sleeve+gastrectomy+is+effective+in+two+genetic+mouse+models+of+glucagon-like+Peptide+1+receptor+deficiency%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Vertical sleeve gastrectomy is effective in two genetic mouse models of glucagon-like Peptide 1 receptor deficiency.</a> Diabetes. 62 (7), 2380-2385 (2013).</li>
<li>Pressler, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vertical+sleeve+gastrectomy+restores+glucose+homeostasis+in+apolipoprotein+A-IV+KO+mice%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Vertical sleeve gastrectomy restores glucose homeostasis in apolipoprotein A-IV KO mice.</a> Diabetes. 64 (2), 498-507 (2015).</li>
</ol>

The authors declare no conflicts of interest, except Dr. Cummings received funding from Eli Lilly and Company.

Bariatric surgery is the most effective long-term treatment for obesity and results in other health benefits such as high rates of type 2 diabetes and hypertension remission1,9,15. Murine models of bariatric surgery provide a powerful tool with which to identify the mechanisms by which bariatric surgery causes rapid and pronounced improvements in obesity comorbidities. Furthermore, murine models of bariatric surgery provide a no...

As the obesity epidemic continues to grow worldwide bariatric surgery has gained popularity as it is the most effective long-term treatment for obesity1. Unfortunately, weight loss by diet and exercise is difficult to achieve and relatively ineffective over the long-term2,3. Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is defined as the manipulation of the gastrointestinal tract for the purpose of weight loss1,4. Although weight loss is a prominent outcome of bariatric surgery, bariatric surgery provides other health benefits such as improving obesity comorbidities and extending lifespan5. For example, bariatric surgery results in high rates of remission of type 2 diabetes and hypertension, and reductions in the lifetime risk of the development of certain types of cancer1,6,7. Of note, the effect of bariatric surgery causing remission of type 2 diabetes and hypertension is often observed soon after surgery and prior to weight loss8,9. This highlights the concept that there are mechanisms independent of body weight contributing to the health benefits observed after surgery. Animal models of bariatric surgery have been developed and are utilized to study the mechanisms by which these health benefits occur10,11,12.
We have validated a mouse model of VSG, which we have applied to various genetically modified mouse models to study the mechanisms by which bariatric surgery improves obesity comorbidities such as type 2 diabetes, hypertension, and colorectal cancer10,11,12. Rodent models allow more experimental control and the ability to perform genetic or pharmaceutical manipulation to define the role of specific genes or signaling pathways of interest. We are focusing primarily on VSG because VSG is the most commonly performed bariatric procedure in the clinic in the United States13. Additionally, VSG is a simple surgical model with fewer anatomic modifications compared to other procedures such as Roux-en-Y gastric bypass or biliopancreatic diversion.
Our mouse model of VSG recapitulates the following effects of bariatric surgery observed in humans: weight loss, reduced food intake, improved glucose regulation, improved islet function, increased post-prandial glucagon-like peptide-1 (GLP-1) secretion, reduced arterial blood pressure, and increased circulating bile acid concentrations10,11,12,13,14,15. Therefore, this is an ideal model to study the body weight dependent and independent mechanisms by which VSG improves or resolves obesity comorbidities. In addition, it is a reliable model that can be combined with other surgical procedures, allowing for the investigation of the impact of VSG under various disease conditions with greater mechanistic insight12.

<table><tbody><tr><td>45% high fat diet</td><td>Research Diets</td><td>D12451</td><td></td></tr><tr><td>60% high fat diet</td><td>Research Diets</td><td>D12492</td><td></td></tr><tr><td>Boost</td><td>Nestle</td><td>160-67538</td><td>rich chocolate flavor</td></tr><tr><td>6-0 Suture</td><td>Ethicon</td><td>Z432</td><td>monofilament absorbable/taper</td></tr><tr><td>7-0 Suture</td><td>Covidien</td><td>8866127-01</td><td>monofilament absorbable/taper</td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>23-400-118</td><td>small</td></tr><tr><td>Cotton Swabs</td><td>Fisherbrand</td><td>233-400-101</td><td>large</td></tr><tr><td>Gauze</td><td>Various</td><td></td><td>4x4 4 ply and 2x2 4 ply</td></tr><tr><td>Foil</td><td>Various</td><td></td><td></td></tr><tr><td>Surgery drape</td><td>Various</td><td></td><td></td></tr><tr><td>0.9% saline solution</td><td>Various</td><td></td><td></td></tr><tr><td>LRS</td><td>Hospira</td><td>170RX</td><td></td></tr><tr><td>Betadine</td><td>Various</td><td></td><td></td></tr><tr><td>Alcohol</td><td>Various</td><td></td><td></td></tr><tr><td>Eye Ointment</td><td>Paralube&amp;reg; Vet Ointment</td><td>17033-211-38</td><td></td></tr><tr><td>Tissue Adhesive</td><td>Vetbond</td><td>1469SB</td><td></td></tr><tr><td>Meloxicam (Metacam)</td><td>Boehringer Ingelheim</td><td>141-213</td><td>5 mg/ml</td></tr><tr><td>Enrofloxacin</td><td>Baytril</td><td>08713254-186599</td><td>22.7 mg/ml</td></tr><tr><td>Thin tipped hemostats</td><td>Fine Science Tools</td><td>13021-12</td><td></td></tr><tr><td>Metzenbaum Scissor</td><td>Fine Science Tools</td><td>14018-18</td><td></td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Dumont Forcep</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Serrated Forcep</td><td>Fine Science Tools</td><td>11020-12</td><td></td></tr><tr><td>Gavage needle</td><td>Fine Science Tools</td><td>18060-20</td><td></td></tr><tr><td>Microneedle driver</td><td>Fine Science Tools</td><td>12075-14</td><td></td></tr><tr><td>Spring Scissor</td><td>Fine Science Tools</td><td>15396-00</td><td></td></tr><tr><td>Insulin syringe</td><td>Various</td><td></td><td></td></tr><tr><td>1mL syringe</td><td>Various</td><td></td><td></td></tr><tr><td>20mL syringe</td><td>Various</td><td></td><td></td></tr><tr><td>Glucometer (one touch ultra mini)</td><td>Lifescan</td><td>70021208</td><td></td></tr><tr><td>Multiplex insulin and GLP-1 kit</td><td>Meso Scale Discovery</td><td>K15171C-1</td><td></td></tr><tr><td>GraphPad Prism 6.00</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>Nestlets</td><td>Ancare</td><td>NES3600</td><td></td></tr></tbody></table>

a murine model of vertical sleeve gastrectomy

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. Bariatric surgery is currently the most effective long-term treatment for obesity. In addition to weight loss, bariatric surgery produces additional health benefits such as remission of type 2 diabetes, remission of hypertension, and decreased risk of developing certain types of cancer. The mechanisms beyond weight loss for these benefits remain incompletely defined. Therefore, animal models of bariatric surgery are being developed and validated to identify the mechanisms leading to these benefits, with the goal of improving understanding of gastrointestinal physiology and identifying new therapeutic targets. VSG has become the most commonly performed bariatric procedure in the clinic in the United States because it is highly effective at producing weight loss and metabolic improvement, and is simpler to perform than other bariatric procedures. Therefore, we have developed and validated a murine model of VSG. This murine VSG model recapitulates many of the effects of VSG seen in humans, including improved glucose and blood pressure regulation. The method is based on isolation of the stomach, ligation of gastric vessels, and removal of 70% of the stomach by transecting along the greater curvature of the stomach. We have successfully applied this surgical protocol to various genetically modified mouse lines to define the mechanistic contributors to the benefits of VSG. Furthermore, this murine VSG model has been combined with other surgical techniques, to achieve deeper mechanistic insight. Therefore, this is a simple and versatile model for studying gastrointestinal physiology and the health benefits of bariatric surgery.

The sham and VSG procedures are depicted in Figure 1. Figure 1A shows where the suture line is placed along the gastric walls during the sham procedure. This same area is where the stomach is cut during VSG surgery. Figure 1B shows the tubular remnant of stomach left after performance of VSG.
Statistics and Data Analysis<br...

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A Murine Model of Vertical Sleeve Gastrectomy

The following describes the performance of vertical sleeve gastrectomy in mice. This is a type of weight-loss surgery that involves removal of approximately 70% of the stomach.

Bariatric surgery, such as vertical sleeve gastrectomy (VSG), is a surgery of the gastrointestinal tract that is performed for the purpose of weight loss. ...

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11.1K Views.  Cornell University. Het volgende beschrijft de prestaties van verticale sleeve Gastrectomie in muizen. Dit is een soort gewichtsverlies chirurgie dat verwijdering van ongeveer 70% van de maag.

Video: A Murine Model of Vertical Sleeve Gastrectomy

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

Techniques of Sleeve Gastrectomy and Modified Roux-en-Y Gastric Bypass in Mice

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is frequently associated with metabolic complications, such as type 2 diabetes, hypertension, and dyslipidemia, reducing life expectancy and quality. In the absence of any effective noninvasive treatments, bariatric surgery is a valuable therapeutic option for patients with morbid obesity (body mass index (BMI) &#62;40 kg/m2), leading to long-term, sustained weight loss and improvements in metabolic complications. However, the underlying cellular and molecular mechanisms sustaining the beneficial effects of bariatric surgery are not yet fully understood. Due to the numerous genetically-modified strains available, the mouse model is the most convenient animal model to explore the molecular mechanisms behind the pleiotropic beneficial effects of bariatric surgeries. Here, we detailed the optimized healthcare methods and surgical protocols in mice for the two most widely-used bariatric surgeries: the sleeve gastrectomy and the modified Roux-en-Y gastric bypass. Deciphering the molecular mechanisms underlying the therapeutic effects of bariatric surgeries offers the promise of identifying new therapeutics targets.

Bariatric surgery is the most efficient way to reduce body weight and the deadly metabolic complications (diabetes, obesity, and dyslipidemia) frequently associated with morbid obesity. Mouse models of bariatric surgery represent a unique asset for deciphering molecular mechanisms behind the beneficial effects of these surgeries on diabetes, hypertension, and dyslipidemia.

Obesity is a major public health issue, with a prevalence of 4 to 28% for men and 6.2 to 36.5% for women in Europe (from 2003 to 2008). Morbid obesity is ...

Medicine

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Sleeve Gastrectomy in Mice using Surgical Clips

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 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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Single-Anastomosis Duodeno-Ileal Bypass with Sleeve Gastrectomy Model in Mice

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes mellitus, dyslipidemia, non-alcoholic steatohepatitis, cardiovascular events, and cancers) through restrictive and malabsorptive mechanisms. Understanding the mechanisms by which these procedures allow such improvements often require their transposition into animals, especially in mice, because of the ease of generating genetically modified animals. Recently, the single-anastomosis duodeno-ileal bypass with sleeve gastrectomy (SADI-S) has emerged as a procedure that uses both restrictive and malabsorptive effects, which is being used as an alternative to gastric bypass in case of major obesity. Thus far, this procedure has been associated with strong metabolic improvements, which has led to a marked increase in its use in daily clinical practice. However, the mechanisms underlying these metabolic effects have been poorly studied as a result of a lack of animal models. In this article, we present a reliable and reproducible model of SADI-S in mice, with a special focus on perioperative management. The description and use of this new rodent model will be helpful for the scientific community to better understand the molecular, metabolic, and structural changes induced by the SADI-S and to better define the surgical indications for clinical practice.

Single-anastomosis duodeno-ileal bypass (SADI-S) is an emerging bariatric procedure with important metabolic effects. In this article, we present a reliable and reproducible model of SADI-S in mice.

Obesity is a major health issue worldwide. As a response, bariatric surgeries have emerged to treat obesity and its related comorbidities (e.g., diabetes ...

Murine Vertical Sleeve Gastrectomy: A Surgical Procedure to Downsize Stomach in Experimental Mouse Models

Source: Garibay, D., Cummings, B. P. <a href="https://www.jove.com/video/56534" target="_blank">A Murine Model of Vertical Sleeve Gastrectomy.</a> J. Vis. Exp. (2017)
In this video, a vertical sleeve gastrectomy procedure is performed on an experimental mouse model. This procedure helps remove a significant portion of the stomach along its greater curvature.

Source: Garibay, D., Cummings, B. P. A Murine Model of Vertical Sleeve Gastrectomy. J. Vis. Exp. (2017)
In this video, a vertical sleeve gastrectomy ...

A Murine Model of Vertical Sleeve Gastrectomy

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