Name: a protocol for roux-en-y gastric bypass in rats using linear staplers
Uploaded: 2021-08-21T15:00:00.000+00:00
Duration: 11 min 58 s
Description: Watch this Scientific Journal Video about A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers at JoVE.com

This study was funded by the American Society for Metabolic and Bariatric Surgery Research Award. Ethicon graciously supplied sutures, staplers, and clips. The lead author&#39;s doctoral research was funded in by the University of Alberta Clinician Investigator Program and the Alberta Innovates Clinician Fellowship. We would also like to thank Michelle Tran for her medical illustration of the RYGB anatomy.

Animal use protocols were approved by the Health Science Animal Care and Use Committee at the University of Alberta (AUP00003000). See Figure 1 for a diagram demonstrating the RYGB anatomy.
1. Roux-en-Y gastric bypass
<ol>
	<li>Preparation of animals and operative setup
	<ol>
		<li>One week prior to the surgery, provide the rats with oral rehydration therapy and liquid diet in addition to their solid diet and water to acclimatize them to this new diet.</li>
		<li>Fast rats with only access to water for 12-18 h prior to the surgery.
		<ol>
			<li>Ensure rats are fasted on a raised wire platform so that they cannot consume bedding material.</li>
		</ol>
		</li>
		<li>Inject rats with subcutaneous long buprenorphine sustained release (SR) at a dose of 1 mg/kg immediately before surgery.</li>
		<li>Autoclave all surgical instruments, towels, and drapes.</li>
		<li>Clean the operating surface, heating pad, and anesthetic nose cone with 70% ethanol.</li>
		<li>Set up the operating surface with an operating microscope, anesthetic machine, and supplies in a manner that is ergonomic for the operating surgeon.</li>
		<li>Use a temperature-regulated heating pad and set to 37 &#176;C.</li>
		<li>Place a sterile drape or towel over the heating pad.</li>
		<li>Fill a 50 mL sterile conical tube with 0.9% saline.</li>
	</ol>
	</li>
	<li>Anesthetic induction and preparation
	<ol>
		<li>Induce anesthesia using 4% isoflurane as per previously established protocols15.</li>
		<li>Apply pressure to the hindfoot of all four limbs to ensure there is no pain response.</li>
		<li>Check for adequate anesthesia and respiratory rate after every 5 min.</li>
		<li>Apply lubricant to both eyes to prevent drying.</li>
		<li>Shave hair from the abdomen.</li>
		<li>Clean the abdomen with a povidone-iodine solution. Allow the solution to dry, and change into sterile gloves.</li>
		<li>Drape the rat with an opening in the drape to expose the abdomen.</li>
		<li>Place instruments, sutures, cotton swabs, and a 10 mL syringe in a location that permits easy access during the procedure.</li>
	</ol>
	</li>
	<li>Median laparotomy
	<ol>
		<li>Make a 3 cm incision in the upper midline of the abdomen using a scalpel, just below the xyphoid process as a landmark.</li>
		<li>Using scissors, divide the fascia and peritoneum, with care to stay midline on the linea alba to reduce bleeding from the rectus abdominus. If there is bleeding, control it with thermal or electrocautery.</li>
	</ol>
	</li>
	<li>Mobilizing the stomach
	<ol>
		<li>Using two wet cotton swabs, bluntly dissect gastric attachments.</li>
		<li>When encountering dense adhesions, use thermal cautery to divide gastric attachments with care to avoid cauterizing the stomach. Sharply divide the ligament between the stomach and the accessory liver lobe to reduce the risk of liver tearing with stomach mobilization.</li>
		<li>For larger blood vessels, especially at the short gastric arteries, ligate using 6-0 polypropylene suture.</li>
		<li>Create a window on the right distal side of the esophagus but proximal to the left gastric artery. Ensure that a cotton swab can reach into this area posteriorly. The stomach is adequately mobilized when it can be exteriorized outside of the abdomen.</li>
	</ol>
	</li>
	<li>Identify and divide the jejunum
	<ol>
		<li>Identify the ligament of Treitz by following the jejunum proximally until observing it is attachment to the transverse mesocolon.</li>
		<li>Measure 7 cm distally, identify a location between mesenteric vessels, and divide the bowel with micro scissors. Avoid Peyer&#39;s patches when dividing the bowel. Take care to only divide the bowel and not the mesentery.</li>
		<li>Place a clean, saline soaked sponge prior to dividing the bowel to minimize contamination.</li>
		<li>Check for the presence of a small crossing vessel in the mesentery at the border of the small bowel and divide this with cautery to avoid bleeding.</li>
		<li>Continue to divide the mesentery 1 cm towards the mesenteric base.</li>
		<li>Identify the proximal and distal jejunum. Place the proximal jejunum under a wet gauze on the rat&#39;s right and the distal jejunum on the rat&#39;s left.</li>
	</ol>
	</li>
	<li>Stapling the stomach
	<ol>
		<li>Insert a 45 mm linear cutting stapler with 3.5 mm staple height across the white line of the forestomach to create a smaller pouch. Wait for 10 s before firing the stapler.</li>
		<li>Place pressure using gauze on the staple lines for 1 min to ensure hemostasis. If hemostasis is not achieved with pressure alone, bleeding along the staple line is oversewn using 6-0 polypropylene figure of eight sutures.</li>
		<li>Perform a second staple fire across the stomach into the window created previously. Wait for 10 s before firing the stapler. Pressure is held along the staple line to ensure hemostasis, and oversewing may be needed.</li>
	</ol>
	</li>
	<li>Gastrojejunostomy
	<ol>
		<li>Make a gastrotomy immediately after stapling the stomach. Delays in this can cause gastric distension and aspiration as the stomach is discontinuous after the second gastric staple.</li>
		<li>Using an 11-blade scalpel, create a gastrotomy at the distal pouch. Express gastric contents through the gastrotomy. This is important to prevent gastric distension and aspiration. Lengthen this gastrotomy using micro scissors to approximately 5 mm. The gastrotomy is made large enough for the cotton swab tip just to fit through.</li>
		<li>Mobilize the distal end of the jejunum adjacent to the gastrotomy and place such that the mesentery is not twisted.</li>
		<li>While suturing the anastomosis, ensure that the bowel is kept moist by covering it with saline-soaked gauze and reapplying saline regularly.</li>
		<li>Using 6-0 polydioxanone or polypropylene suture, place a stay suture at the inferior margin of the anastomosis and gently retract using a snap. Tie with three knots.</li>
		<li>Place a stay suture at the superior margin of the anastomosis and gently retract using a snap. Tie with six knots.</li>
		<li>Suture the anterior side of the anastomosis in a continuous fashion, taking bites 1 mm wide and 1 mm apart with care to avoid taking the backside.</li>
		<li>Once the suture has reached the inferior stay suture, tie these together with an additional six knots.</li>
		<li>Once the anterior side is complete, flip the bowel and stomach over and pass the inferior stay suture through the mesenteric defect. Reapply the snap and retract inferiorly.</li>
		<li>For the posterior side of the anastomosis, place full thickness interrupted 6-0 sutures, 1 mm wide, and spaced 1 mm apart, with care to avoid taking the backside. These are tied with six knots each. 
		NOTE: The anterior side of the anastomosis is sutured in a continuous fashion while the posterior side is done in an interrupted fashion. This prevents potential stricture or stenosis associated with a continuous circumferential closure.</li>
		<li>Check for the leakage by gently pushing luminal contents across the anastomosis. If there are areas with leakage, carefully reinforce them with interrupted sutures. Take care to avoid taking the backwall when reinforcing with extra sutures.</li>
	</ol>
	</li>
	<li>Jejunojejunostomy
	<ol>
		<li>From the gastrojejunostomy, measure 20 cm distally.</li>
		<li>Create a jejunotomy on the antimesenteric side using the 11-blade scalpel. Avoid making the jejunotomy over Peyer&#39;s patches.</li>
		<li>Extend this jejunotomy using micro scissors, such that it is the same size as the biliopancreatic limb. Ensure that a cotton swab just fits inside.</li>
		<li>Place the biliopancreatic limb such that there is no twisting of the mesentery.</li>
		<li>Perform the anastomosis similarly to the gastrojejunostomy with 6-0 stay sutures on the superior and inferior sides. The anterior side is performed with continuous sutures while the posterior side is performed with interrupted sutures.</li>
		<li>Ensure that the bowel is kept moist with saline during this anastomosis.</li>
		<li>Check for leakage by gently pushing luminal contents through the anastomosis. If there are areas with leakage, reinforce them with interrupted sutures.</li>
	</ol>
	</li>
	<li>Reposition the bowel and stomach
	<ol>
		<li>Ensure that there is no twisting of the pouch, remnant stomach, or liver. Ensure that the left lobe of the liver is anterior to the stomach and not trapped behind the pouch as this can cause compressive liver ischemia.</li>
		<li>Position the bowel in the abdomen in its natural position such that there is no twisting.</li>
	</ol>
	</li>
	<li>Abdominal closure
	<ol>
		<li>Close the fascia with 3-0 polyglactin in a continuous fashion. 3-0 polydioxanone may also be used.</li>
		<li>Close the skin with 2-0 silk in a continuous fashion.</li>
	</ol>
	</li>
	<li>Anesthetic emergence
	<ol>
		<li>Decrease isoflurane to zero but continue supplemental oxygen.</li>
		<li>Administer a local anesthetic as a splash block to the incision.</li>
		<li>Administer 10 mL of subcutaneous 5% dextrose in normal saline (D5NS) in the subcutaneous tissue behind the neck.</li>
		<li>Place an Elizabethan rat collar before the rat is fully awake. Take care to fit it snugly but not too tight to cause discomfort. 
		&#8203;NOTE: The collar is kept on until day 5 to prevent wound dehiscence.</li>
	</ol>
	</li>
</ol>
2. Sham surgery
NOTE: Sham surgery is performed similar to RYGB, however, no anastomoses are performed.
<ol>
	<li>A gastrotomy is created and then closed with 6-0 polydioxanone or polypropylene sutures.</li>
	<li>A jejunotomy is created 7 cm distal to the ligament of Treitz and then closed with 6-0 polydioxanone or polypropylene sutures.</li>
</ol>
3. Postoperative care
<ol>
	<li>Postoperative care
	<ol>
		<li>House rats individually and keep them on raised wire platforms until solid food is reintroduced to prevent consumption of bedding and luminal obstruction. 
		NOTE: Postoperative diet is resumed gradually as edema at the gastrojejunostomy can cause obstruction with the early resumption of solid diet.</li>
		<li>Inspect the feet daily while rats are on raised wire platforms for any skin changes.</li>
		<li>Keep rats on water and oral rehydration therapy diet for the first 72 h.</li>
		<li>Administer 10 mL of D5NS every 12 h for the first 72 h.</li>
		<li>Administer subcutaneous short-acting buprenorphine at 0.01 mg/kg if rats appear to be in pain. The Rat Grimace Scale is used to assess for pain16.</li>
		<li>On postoperative day 3, add rodent liquid diet. Continue to provide water and oral rehydration therapy.</li>
		<li>On postoperative day 5, restart high-fat diet. Continue to provide water and liquid diet. Remove the Elizabethan collar.</li>
		<li>On postoperative day 7, discontinue liquid diet.</li>
		<li>Remove skin sutures on postoperative day 10-14.</li>
	</ol>
	</li>
</ol>

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Ethicon supplied two 45 mm linear cutting staplers, multiple 3.5 mm stapler reloads, and 6-0 polypropylene sutures. Authors have no other conflicts of interest to declare.

RYGB involves the creation of a small gastric pouch (less than 30 mL), and the creation of a biliopancreatic limb and a Roux limb (Figure 1). In humans, the biliopancreatic limb is typically 30 to 50 cm and transports secretions from the gastric remnant, liver, and pancreas. The Roux limb is typically 75 to 150 cm in length and is the primary channel for ingested food. The common channel is the remaining small bowel distal to where the two limbs join and is where the majority of digestion an...

Obesity and type 2 diabetes have become worldwide epidemics1. Although medical weight loss can improve diabetes in patients, those with severe diabetes benefit most from bariatric surgery. Bariatric surgery has proven to be safe and effective at weight loss and improving or curing type 2 diabetes2,3, even in those with long-standing disease4. Metabolic bariatric procedures, such as the current gold-standard Roux-en-Y gastric bypass (RYGB) surgery, induce rapid and sustained improvements in glucose homeostasis while also reducing the need for diabetic medications5,6,7.
After RYGB, glucose homeostasis improvement occurs rapidly and is independent of the weight loss8. Two major theories have been proposed to explain the metabolic changes associated with diabetes remission that occur following metabolic surgery. First, the hindgut hypothesis postulates that, after bypass, higher concentrations of undigested nutrients reach the distal intestine enhancing the release of hormones such as GLP-1. On the other hand, the foregut hypothesis suggests that bypassing the proximal intestine reduces the secretion of anti-incretin hormones. Both of these effects could lead to early improvement of glucose metabolism9.
Animal models have the potential to be a powerful tool to study these mechanisms. However, a major barrier in utilizing mouse or rat models is the technical difficulty in performing these procedures. Most studies have relied on mouse or rat models10,11,12. Mouse models have been difficult as the mouse stomach is too small to use stapler devices11, and mortality rates are unacceptably high, ranging from 17 to 52%13. In rats, some protocols remain technically difficult to perform due to complex ligation of gastric vessels prior to dividing the stomach12,14. Other models divide the stomach using a stapler but leave a large pouch not consistent with the post RYGB human anatomy11. In this model, we provide detailed instructions on how to perform RYGB using linear staplers in a rat model resulting in a gastric pouch more in keeping with that of human anatomy. Overall, this procedure was associated with excellent survival rates and metabolic outcomes.

<table><tbody><tr><td>2-0 Silk Sutures</td><td>Ethicon</td><td>K533</td><td></td></tr><tr><td>3-0 Vicryl Sutures</td><td>Ethicon</td><td>J219H</td><td></td></tr><tr><td>4% Isoflurane</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>5% Dextrose and 0.9% Sodium Chloride Solution - 1000 mL</td><td>Baxter</td><td>2B1064</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>6-0 Prolene Sutures</td><td>Ethicon</td><td>8805H</td><td></td></tr><tr><td>Anesthetic Machine</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Animal Hair Shaver</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Betadine Solution</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Castrojievo Needle Holder with lock 14 cm (smooth curved)</td><td>World Precision Instruments</td><td>503258</td><td></td></tr><tr><td>ECHELON FLEX Articulating Endoscopic Linear Cutter</td><td>Ethicon</td><td>EC45A</td><td></td></tr><tr><td>Economy Tweezers #4</td><td>World Precision Instruments</td><td>501978</td><td></td></tr><tr><td>ENDOPATH ETS Articulating Linear Cutter 45mm Reloads</td><td>Ethicon</td><td>6R45B</td><td></td></tr><tr><td>Far Infrared Warming Pad Controller with warming pad (15.2 cm W x 20.3 cm L), pad temperature probe, and 10 disposable, non-sterile sleeve protectors</td><td>Kent Scientific</td><td>RT-0515</td><td></td></tr><tr><td>Large Rat Elizabethan Collar</td><td>Kent Scientific</td><td>EC404VL-10</td><td></td></tr><tr><td>Liquid Diet Feeding Tube (150 mL)</td><td>Bio-Serv</td><td>9007</td><td></td></tr><tr><td>Liquid Diet Feeding Tube Holder (short adjustable)</td><td>Bio-Serv</td><td>9015</td><td></td></tr><tr><td>Micro Mosquito Forceps</td><td>World Precision Instruments</td><td>500452</td><td></td></tr><tr><td>Micro Scissors</td><td>World Precision Instruments</td><td>503365</td><td></td></tr><tr><td>Mouse Diet, High Fat Fat Calories (60%), Soft Pellets</td><td>Bio-Serv</td><td>S3282</td><td></td></tr><tr><td>No. 11 Blade and Scalpel Handle</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>OPMI Vario Surgical Microscope</td><td>ZEISS</td><td>S88</td><td></td></tr><tr><td>Raised Floor Grid</td><td>Tecniplast</td><td>GM500150 Raised Floor Grid</td><td></td></tr><tr><td>Rodent Liquid Diet, Lieber-DeCarli &#039;82, Control, 4 Liters/Bag</td><td>Bio-Serv</td><td>F1259</td><td></td></tr><tr><td>Sodium Chloride Irrigation 0.9% Solution - 500 mL</td><td>Baxter</td><td>JF7633</td><td></td></tr><tr><td>Sterile Cotton Swabs</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Sterile Drape</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Sterile Towel</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Thermal Cautery Unit</td><td>World Precision Instruments</td><td>501293</td><td></td></tr></tbody></table>

a protocol for roux-en-y gastric bypass in rats using linear staplers

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and metabolic changes are not well understood. Multiple factors are thought to play a role, including reduced caloric intake, decreased nutrient absorption, increased satiety, the release of satiety-promoting hormones, shifts in bile acid metabolism, and alterations in the gut microbiota.
The rat RYGB model presents an ideal framework to study these mechanisms. Prior work on mouse models have had high mortality rates, ranging from 17 to 52%, limiting their adoption. Rat models demonstrate more physiologic reserve to surgical stimulus and are technically easier to adopt as they allow for the use of surgical staplers. One challenge with surgical staplers, however, is that they often leave a large gastric pouch which is not representative of RYGB in humans. 
In this protocol, we present a RYGB protocol in rats that result in a small gastric pouch using surgical staplers. Utilizing two stapler fires which remove the forestomach of the rat, we obtain a smaller gastric pouch similar to that following a typical human RYGB. Surgical stapling also results in better hemostasis than sharp division. Additionally, the forestomach of the rat does not contain any glands and its removal should not alter the physiology of RYGB.
Weight loss and metabolic changes in the RYGB cohort were significant compared to the sham cohort, with significantly lower glucose tolerance at 14 weeks. Furthermore, this protocol has an excellent survival of 88.9% after RYGB. The skills described in this protocol can be acquired without previous microsurgical experience. Once mastered, this procedure will provide a reproducible tool for studying the mechanisms and effects of RYGB.

Animals and housing 
36 male Wistar rats were housed in pairs and were fed 60% sterile rodent high-fat diet starting from six weeks of age (Figure 2). At 16 weeks of age, they underwent RYGB or sham surgery. After the first postoperative week, rats were resumed on a high fat diet. Half of the rats were euthanized at 2 weeks post-operative and the other half were euthanized at 14 weeks postoperative.
Mortality 
Overall, 33 (9...

Watch this Scientific Journal Video about A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers at JoVE.com

A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers

Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

Roux-en-Y gastric bypass (RYGB) is commonly performed for the treatment of severe obesity and type 2 diabetes. However, the mechanism of weight loss and ...

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Medicine

5.3K Views.  University of Alberta. Roux-en-Y gastric bypass (RYGB) is performed to treat obesity and diabetes. However, the mechanisms underlying RYGB's efficacy are not fully understood, and studies are limited by technical difficulty leading to high mortality in animal models. This article provides instructions on how to perform RYGB in rats with high success rates.

Video: A Protocol for Roux-en-Y Gastric Bypass in Rats using Linear Staplers

Roux-en-Y Gastric Bypass Operation in Rats

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality benefit is bariatric surgery1,2. Consequently, there has been a steady rise in the number of bariatric operations done worldwide in recent years with the Roux-en-Y gastric bypass (gastric bypass) being the most commonly performed operation3. Against this background, it is important to understand the physiological mechanisms by which gastric bypass induces and maintains body weight loss. These mechanisms are yet not fully understood, but may include reduced hunger and increased satiation4,5, increased energy expenditure6,7, altered preference for food high in fat and sugar8,9, altered salt and water handling of the kidney10 as well as alterations in gut microbiota11. Such changes seen after gastric bypass may at least partly stem from how the surgery alters the hormonal milieu because gastric bypass increases the postprandial release of peptide-YY (PYY) and glucagon-like-peptide-1 (GLP-1), hormones that are released by the gut in the presence of nutrients and that reduce eating12.
During the last two decades numerous studies using rats have been carried out to further investigate physiological changes after gastric bypass. The gastric bypass rat model has proven to be a valuable experimental tool not least as it closely mimics the time profile and magnitude of human weight loss, but also allows researchers to control and manipulate critical anatomic and physiologic factors including the use of appropriate controls. Consequently, there is a wide array of rat gastric bypass models available in the literature reviewed elsewhere in more detail 13-15. The description of the exact surgical technique of these models varies widely and differs e.g. in terms of pouch size, limb lengths, and the preservation of the vagal nerve. If reported, mortality rates seem to range from 0 to 35%15. Furthermore, surgery has been carried out almost exclusively in male rats of different strains and ages. Pre- and postoperative diets also varied significantly.
Technical and experimental variations in published gastric bypass rat models complicate the comparison and identification of potential physiological mechanisms involved in gastric bypass. There is no clear evidence that any of these models is superior, but there is an emerging need for standardization of the procedure to achieve consistent and comparable data. This article therefore aims to summarize and discuss technical and experimental details of our previously validated and published gastric bypass rat model.

Numerous studies using gastric bypass rat models have been recently conducted to uncover the underlying physiological mechanisms of Roux-en-Y gastric bypass operations. This article aims to demonstrate and discuss the technical and experimental details of our published gastric bypass rat model to understand advantages and limitations of this experimental tool.

Currently, the most effective therapy for the treatment of morbid obesity to induce significant and maintained body weight loss with a proven mortality ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

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 ...

One-anastomosis Gastric Bypass (OAGB) in Rats

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical model, longitudinal studies can be carried out to provide an improved understanding of the mechanisms underlying the outcomes seen after bariatric surgery in humans. For this purpose, rats are operated on through a laparotomy under general anesthesia with isoflurane. First, the surgeon creates a long and tubular gastric pouch: after greater curve and hiatal dissection, the nonglandular stomach is stapled and removed. Then, the remaining stomach is also stapled in order to create a gastric tube and exclude the antrum of the stomach. After that, the surgeon performs a single end-to-side gastrojejunostomy 35 cm from the duodenojejunal angle. This limb length has been chosen in order to reproduce the same ratio between the biliopancreatic limb (BPL) and common limb (CL) length as in human bariatric surgery. The operation ends by aponeurotic and cutaneous closure. The early postoperative management consists of subcutaneous hydration, an intramuscular prophylactic antibiotic injection, a parietal injection of xylocaine, the administration of painkillers, and a progressive reintroduction of diet.

One-anastomosis Gastric Bypass OAGB in Rats

This protocol is for the performance of one-anastomosis gastric bypass (OAGB) on rat. The operator performs a long and tubular-stapled gastric pouch followed by hand-sewn anastomosis. For this model, the operator reproduces the same ratio between biliopancreatic and common limb in humans; therefore, the biliopancreatic limb measures 35 cm.

The goal of this protocol is to set up a preclinical model of bariatric surgery and, more specifically, OAGB in obese rats. Based on this preclinical ...

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 ...

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 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. ...

Biology

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.

Mysia pionowa rękawowa resekcja żołądka: zabieg chirurgiczny mający na celu zmniejszenie rozmiaru żołądka w eksperymentalnych modelach mysich

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

JoVE Encyclopedia of Experiments

Cancer Research

Gastrointestinal Cancer

Animal model studies

Roux-en-Y Gastric Bypass Procedure: A Bariatric Surgical Procedure for Gastric Resectioning in Murine Model

For a Roux-en-Y gastric bypass, make a midline skin incision from the sternum to the middle of the abdomen, protecting the skin with a sterile compress soaked with 37 degrees Celsius saline solution.
Externalize the intestine and measure 8 centimeters from the pyloris. Using 5-0 non-absorbable sutures, place two ligatures on the intestine on either side of the 8-centimeter mark, and cut the tissue between the sutures.
Then, place the proximal limb of the tissue in the upper left quadrant of the abdomen, and the distal limb facing the alimentary limb, 6 centimeters below the proximal limb. Using micro scissors, cut both the proximal limb and the intestinal loop, and make two anti-mesenteric incisions of the same length. Next, use two 8-0 non-absorbable sutures to make a side-to-side anastomosis; placing the dorsal side anastomosis first, followed by the ventral side anastomosis.
Gently roll two moistened cotton swabs toward each side of the anastomosis, to confirm that the suture is leakproof, and carefully externalizing the stomach as just demonstrated. Place a re-absorbable hemostatic collagen compress behind the organ, and used curved micro forceps to pass a 5-0 non-absorbable suture through the omentum to ligate the pylorus.
Cut the ventral side of the stomach 1.5 centimeters from the pylorus and the distal limb, creating two incisions of the same length, followed by a dorsal to ventral side to side anastomosis with two 8-0 non-absorbable sutures. Confirm that the suture does not leak.
Then, close the muscle layer of the abdominal wall and the skin, with 5-0 non-absorbable sutures, and administer 25 milliliters per kilogram of warm saline solution, via subcutaneous injection.
When the mouse has fully recovered from the anesthesia, place the animal alone in a cage in 30 degrees Celsius incubator for 5 days, with daily iron and vitamin supplementation and analgesia, and free access to gel diet food for the first two days. On the third day after surgery, reintroduce solid food to the animal&#39;s diet.