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>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+effects+of+laparoscopic+sleeve+gastrectomy,+gastric+bypass,+and+adjustable+gastric+banding+on+type+2+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+bariatric+surgery+on+mortality+in+Swedish+obese+subjects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+incidence+and+mortality+after+gastric+bypass+surgery.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+management+of+obesity+and+the+relationship+to+cardiovascular+disease.">Surgical management of obesity and the relationship to cardiovascular disease.</a> Circulation. 127 (8), 945-959 (2013).</li><li>Ahmed, A. 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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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Cell+Glucagon-Like+Peptide-1+Receptor+Contributes+to+Improved+Glucose+Tolerance+After+Vertical+Sleeve+Gastrectomy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGR5+contributes+to+glucoregulatory+improvements+after+vertical+sleeve+gastrectomy+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+sleeve+gastrectomy+reduces+blood+pressure+and+hypothalamic+endoplasmic+reticulum+stress+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+sleeve+gastrectomy+leads+the+U.S.+utilization+of+bariatric+surgery+at+academic+medical+centers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+weight+loss+by+gastric+bypass+surgery+versus+hypocaloric+diet+on+glucose+and+incretin+levels+in+patients+with+type+2+diabetes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Health+benefits+of+gastric+bypass+surgery+after+6+years.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGR5+contributes+to+glucoregulatory+improvements+after+vertical+sleeve+gastrectomy+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvement+in+glucose+metabolism+after+bariatric+surgery:+comparison+of+laparoscopic+Roux-en-Y+gastric+bypass+and+laparoscopic+sleeve+gastrectomy:+a+prospective+randomized+trial.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FXR+is+a+molecular+target+for+the+effects+of+vertical+sleeve+gastrectomy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertical+sleeve+gastrectomy+is+effective+in+two+genetic+mouse+models+of+glucagon-like+Peptide+1+receptor+deficiency.">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. 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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&#174; 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...

Watch this Scientific Journal Video about A Murine Model of Vertical Sleeve Gastrectomy at JoVE.com

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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11.0K Views.  Cornell University. 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.

Video: A Murine Model of Vertical Sleeve Gastrectomy

Murine Pancreatic Islet Isolation

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 ...

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 ...

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 ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease caused by vasoconstriction and remodeling of the small arteries in the lungs. This remodeling leads to increased pulmonary vascular resistance, worsened right ventricular function, and premature death. Currently approved therapies for PAH largely target pulmonary vasodilator pathways; however, recent emerging therapeutic modalities are focused on other novel pathways involved in the pathogenesis of the disease, including right ventricle (RV) remodeling. Imaging techniques that allow longitudinal assessment of novel therapeutics are very useful for determining the efficacy of new drugs in preclinical studies. Noninvasive trans-thoracic echocardiography remains the standard approach to evaluating heart function and is widely used in rodent models. However, echocardiographic evaluation of the RV can be challenging due to its anatomical position and structure. In addition, standardized guidelines are lacking for echocardiography&#160;in preclinical rodent models, making it difficult to carry out a uniform assessment of RV&#160;function across studies in different laboratories. In preclinical studies, the monocrotaline (MCT) injury model in rats is widely used to evaluate drug efficacy for treating PAH. This protocol describes the echocardiographic evaluation of the RV in na&#239;ve and MCT-induced PAH rats.

The present protocol describes the echocardiographic characterization of right ventricular morphology and function in a rat model of pulmonary arterial hypertension.

A Murine Model of Vertical Sleeve Gastrectomy

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Murine Pancreatic Islet Isolation

Murine Model of Hindlimb Ischemia

Murine Renal Transplantation Procedure

LAD-Ligation: A Murine Model of Myocardial Infarction

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

Isolation of Murine Coronary Vascular Smooth Muscle Cells

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Comprehensive Echocardiographic Assessment of Right Ventricle Function in a Rat Model of Pulmonary Arterial Hypertension