A Surgical Rat Model of Human Roux-en-YGastric BypassMichael M. Meguid, M.D., Ph.D., Eduardo J.B. Ramos, M.D., Susumu Suzuki, M.D., Ph.D.,Yuan Xu, M.D., Zachariah M. George, B.Sc., Undurti N. Das, M.D., Karen Hughes, A.A.S.,Robert Quinn, D.V.M., Chung Chen, Ph.D., William Marx, D.O.,Paul R.G. Cunningham, M.D.

Obesity affects 30% of the United States population and its detrimental effects are obesity-relatedmetabolic diseases. For patients refractory to conventional weight loss therapy, gastric bypass surgery isone of the proven methods for inducing a sustained weight loss and reversing the metabolic sequelaeof obesity. To understand the mechanisms of weight loss and the amelioration of related metaboliccomorbid conditions, a reproducible animal model is needed. We report our developmental experiencewith rat models of sequential Roux-en-Y gastric bypass after reproducing the diet-induced obesity thatcharacterizes the hallmarks of human obesity. Four experiments were performed to induce weight reduc-tion through successive modifications: In Experiment 1 a 20% stapled gastric pouch with a 16 cm biliary-pancreatic limb and a 10 cm alimentary limb accomplished sufficient weight loss within 10 days toamelioratemetabolic changes associated with obesity, but the occurrence of gastrogastric fistulas preventedsustained weight loss; in Experiment 2 the model was improved by dividing the stomach to avoidgastrogastric fistula, but again sustained weight loss was not achieved; in Experiment 3 the biliary-pancreatic limb was lengthened from 16 to 30 cm, reducing the common channel to approximately 18cm. Sustained weight loss was achieved for 28 days. In Experiment 4 the model in Experiment 3 wasmodified by dividing the stomach between two rows of staples. Sustained weight loss was observed for67 days. We developed a reproducible rat model of Roux-en-Y gastric bypass. The existence of thismodel opens a new field of research in which to study the metabolic sequelae of obesity and themechanisms of weight loss. ( J GASTROINTEST SURG 2004;8:621–630) � 2004 The Society for Surgeryof the Alimentary Tract

KEY WORDS: RYGB, rat model, diet-induced obese rat, weight loss

From the Surgical Metabolism and Nutrition Laboratory, Neuroscience Program, Department of Surgery, University Hospital, and theDepartment of Animal Care, Upstate Medical University (M.M.M., E.J.B.R., S.S., Y.X., Z.M.G., U.N.D., K.H., R.Q., W.M., P.R.G.C.),Syracuse, New York; and the Department of Statistics, Management Information and Decision, Whitman School of Management, SyracuseUniversity (C.C.), Syracuse, New York.Supported in part by the Hendrick’s Fund, grant NIH/DK 003568, by an educational grant from the Department of Surgery, Upstate MedicalUniversity, and by grants from Ethicon, Cincinnati, Ohio.Reprint requests: Michael M. Meguid, M.D., Ph.D., Department of Surgery, University Hospital, 750 E. Adams St., Syracuse, NY 13210.e-mail: meguidm@upstate.edu

The extensive similarity and homology betweenhuman and rodent genomes make animal models apowerful tool to study similar human conditions. Twoexamples are (1) the diabetic dogs of Banting andBest1 that were treated with insulin and (2) the dem-onstration of normal growth in beagle pups adminis-tered total parenteral nutrition by Dudrick et al.2Conversely, the absence of an animal model to helpunderstand the mechanisms of a pathophysiologicprocess makes more difficult the development ofspecific drug therapy. Thus the availability or the

� 2004 The Society for Surgery of the Alimentary TractPublished by Elsevier Inc.

development of an animal model of human diseaseis essential.Obesity is a metabolic condition the etiology of

which is environmental, social, and genetic3; obesityhas achieved epidemic proportions in the UnitedStates.4–6 An increase in bodyweight is associatedwitha significant increase in obesity-related diseases in-cluding type 2 diabetes, hyperlipidemia, and cardio-vascular diseases.7,8 The adverse clinical outcomes ofthese comorbid conditions are such that a 20% in-crease in body weight above ideal is associated with

1091-255X/04/$—see front matterdoi:10.1016/j.gassur.2004.02.003 621

Journal ofGastrointestinal Surgery622 Meguid et al.

a 20% increase in the mortality rate.9,10 An additionalproblem is that morbid obesity is now appearing inchildren with greater frequency.11The following three main treatment modalities are

currently used to induce weight loss: (1) reducingcaloric intake via dieting; (2) increasing energy outputvia exercise, or a combination of the two; and (3) con-trolling malabsorption through diversion of nutrientsfrom segments of the gastrointestinal tract. Unfortu-nately, given our current lifestyle, achieving andmaintaining such long-lasting weight loss is difficultwith only modification of diet and exercise. Currentlyapproved drugs reduce weight by 5% to 10% in 1year, and most persons regain their weight once ther-apy is discontinued.12Because it is difficult to achieve sustained weight

loss with existing clinical treatment, bariatric opera-tions are becoming one of the most common abdomi-nal surgical procedures performed daily in the UnitedStates.13 Current consensus is that a successful opera-tionhas two components: a small gastric pouch to limitthe amount of food ingested, inducing early satiety,and an element of subclinicalmalabsorption, reducingthe amount of food that can be absorbed. Of the nu-merous operations developed during the past decades,the Roux-en-Y gastric bypass (RYGB) is consideredamong themost successful and safe operations follow-ing which morbidly obese patients usually lose anaverage of 49% to 65% of their initial body weightwithin 2 to 5 years.13–15 Besides the robust weight loss,the surgical procedure ameliorates obesity-related dis-eases (initially identified as syndrome X7 and nowcommonly referred to as “metabolic syndrome”), par-ticularly diabetes15 and hyperlipidemia.9,16Despite the success of bariatric operations, they

are associated with recognized perioperative compli-cation rates ranging from 3% to 20% and mortalityrates ranging from 0.5% to 2.0%.17 Furthermore,there is a subset of morbidly obese patients in whomsurgery is life-threatening and contraindicated. Be-cause current drug treatments for obesity do notachieve a sustained weight loss, one of the most effec-tive treatments is gastric bypass. To understand themechanism of weight loss and the amelioration ofrelated metabolic comorbid conditions, a reproduc-ible experimental animal model is needed. We reportour developmental experience with sequential RYGBrat models after reproducing the diet-induced obesitythat characterizes the nature of human obesity. Ourdata show that this model ameliorates obesity-relatedmetabolic changes and induces sustained weight loss.

MATERIAL AND METHODS

The Committee for the Humane Use of Animalsat the State University of New York Upstate Medical

University approved these studies. Animal care wasin accordance with National Institutes of Healthguidelines.

Common Procedures

Diet-Induced Obesity. The method of Levin et al.18was used to produce diet-induced obesity in rats.Three-week-old male Sprague-Dawley pups (CharlesRiver,Wilmington,MA) were housed in wire holdingcages and fed a coarsely ground high-energy diet(D12266; Research Diets, New Brunswick, NJ) con-sisting of 8% corn oil and 44% sweetened condensedmilk added to 48% Purina Rat Chow (Rat ChowDiet5008; Ralston Purina, St. Louis, MO) for 7 to 12weeks to develop obesity. The high-energy diet pro-vided 4.5 kcal/g, of which 21% of the metabolizableenergy was protein, 31%was fat, and 48%was carbo-hydrate; the latter was 50% sucrose. Ten days beforethe operation, rats on the high-energy diet were alsofed the highly palatable liquid supplement Boost-Plus(Mead Johnson, Evansville, IN), providing 1.52 kcal/ml, of which 16%of themetabolizable energy contentwas protein, 34.0% was fat, and 50% was carbohy-drate, to enhance their weight gain. As a controlgroup, 3-week-old Sprague-Dawley pups were fed adiet of regular chow (Rat Chow Diet 5008; RalstonPurina) for the same period (chow-control).Study Conditions. Rats were in constant study sur-

roundings consisting of 12-hour light/dark cycles(light on from 5 AM to 7 PM), room temperature of26 � 1� C, and 45% relative humidity, and they hadfree access to their diets and tap water.Anesthesia and Postoperative Pain Control. For

preoperative procedures, rats were deprived of foodfor 16 to 18 hours and were anesthetized with a mix-ture of ketamine and xylazine (200 mg:5 mg, 0. 8 ml/kg, intraperitoneally). For euthanasia, decapitationwas performed under brief isoflurane anesthesia(Baxter, Chicago, IL). Buprenorphine hydrochloride(Reckitt Benckiser Pharmaceuticals, Richmond, VA),0.05mg/kg,wasgivento theratspostoperatively, twicea day for 3 days.Surgical Procedures. The rat abdomens were

shaved and prepared with Betadine solution (PurduePharma, LP, Stamford, CT). For the shamoperationsa midline incision was made, and the stomach anddistal esophagus were exposed; the small bowel waslaid out for the same duration required for gastricbypass procedures. The abdomen was closed in layerswith 3-0 polyglactin sutures (Ethicon, Cincinnati,OH). For gastric bypass operations, three series ofexperiments were performed in sequence to developthe RYGB model.

Vol. 8, No. 52004 Roux-en-Y Gastric Bypass Model in Diet-Induced Obese Rats 623

Experiment 1: 20% Stapled Gastric PouchWith a16 cm Biliary-Pancreatic Limb, a 10 cm AlimentaryLimb, and a Common Channel of Approximately34 cm. The original procedure, whichwas carried outin obese Zucker rats, was previously described indetail.19Thirty Sprague-Dawleypups (startingweight54.9 � 1.8 g) were studied: 24 diet-induced obese ratsand six chow-fed control rats. Diet-induced obeserats fed for7weekswereassigned tooneof threeopera-tive groups as follows: (1) RYGB; (2) sham-operatedobese (SO-obese), or (3) sham-operated pair-fed. Inthe pair-fed group, food intake was restricted andlinked to the daily food intake consumedby theRYGBrats. The remaining six rats on the chow diet served asgeneral control subjects for measuring and assessingthe difference in body weight throughout this study.Following anesthesia, two double rows of titaniumstaple lines (TRH30-4.8; Ethicon) were placed fromthe lesser curvature, 2 to 3mmbelow the gastroesoph-ageal junction, to the greater curvature, constructing a∼20% gastric pouch preserving the vagus nerve andwithout transecting the stomach (Fig. 1, A). The gas-tric bypass staple line was reinforced with multiple in-terrupted 5-0 polyglactin sutures. The jejunum wasdivided 16 cm below the ligament of Treitz, creatinga 16 cm biliary-pancreatic limb. A 4 to 5 mm end-to-side gastrojejunostomy was sewn using inter-rupted 5-0 polyglactin sutures on the anterior surfaceof the gastric fundus. The stump of proximal jejunumwas closed with a running suture. A 7 to 8 mm side-to-side jejunojejunostomy was sewn 10 cm below thegastrojejunostomy.Thecommonchannelwasapprox-imately 34 cm, as indicated in Fig. 1,A. The procedurelasted approximately 50minutes and the abdomenwasclosed in layers. Body weight and operative complica-tionswereevaluateddaily.RatsdrankwaterandBoost-Plus starting 24 hours after the operation and for thefirst 3 days. This was followed by a solid diet. Forthe first three postoperative days, rats were hydratedwithnormal salinesolution (20ml) injectedsubcutane-ously to prevent dehydration. All of the rats startedto lose weight. However, failure to continue to loseweight occurred in three rats by day 10 (which rep-resents approximately one human year), at which timeall rats were euthanized.Adipose Tissue and Biochemical Studies. Subcuta-

neous abdominal fat and mesenteric, retroperitoneal,and epididymal fat pads were dissected out andweighed. Differences in fat weights were calculated.Adipose tissue samples were collected from identicalanatomic areas in each rat to ensure sampling consis-tency.Bloodwas collected tomeasure glucose, insulin,lipid profile, and leptin. Blood samples for serum andEDTA-rinsed tubes (plasma) were centrifuged for 10minutes at 4� C. The plasma and serum samples were

Fig. 1. RYGB. A, Two double rows of titanium staple lineswere placed without transecting the stomach. B, To overcomethe occasional problem of gastrogastric fistula, the stomachwas divided and both gastric ends were oversewn.

stored at �80� C before analysis. Plasma glucose, tri-glyceride, cholesterol (Sigma, St.Louis,MO), and freefatty acid (WAKO, Richmond, VA) concentrationswere measured by means of enzymatic colorimetrickits. Plasma insulin (ALPCO, Windham, NH) andleptin (DSL, Chicago, IL) concentrations were mea-sured using enzyme immunoassay kits.

Journal ofGastrointestinal Surgery624 Meguid et al.

Experiment 2: 20%DividedGastric PouchWith a16 cm Biliary-Pancreatic Limb, a 10 cm AlimentaryLimb, and a Common Channel of Approximately34 cm. Thispilot studywas performed to establish thefeasibility of dividing the stomach. Twelve diet-induced Sprague-Dawley rats (449.9 � 12.4 g) wereequally divided into RYGB and control groups. Thestomach was divided as shown in Fig. 1, B, to over-come thepreviousgastrogastricfistula.The leftgastricvessels and the vagus were preserved as described ear-lier. Both gastric ends were oversewn using a running5-0 polyglactin suture, and the suture lines were em-brocated. The rest of the procedure was the same asthat described for Experiment 1. This procedure ex-tended our operative time to 120 minutes. Rats drankwater andBoost-Plus starting 24hours after operationfor 7 days, followed by solid diet. Rats were hydratedwith normal saline solution as described earlier. Ratswere euthanized 21 days after the surgical procedurewhen they started to gainweight. Bodyweight and op-erative complications were evaluated daily.Experiment 3: 20% Divided Gastric Pouch, With

a30cmBiliary-PancreaticLimb,a10cmAlimentaryLimb, and a Common Channel of Approximately18 cm. Thirty-six 3-week-old Sprague-Dawley pups(47.3 � 0.5g) were fed for 12 weeks to induce obesity.These obese rats were stratified according to bodyweight and randomly assigned within each stratumto one of three operative groups: (1) RYGB (541.1 �6.7 g); (2) SO-obese (545.0 � 8.0 g); or (3) pair-fed(546.1 � 7.6 g). A single intramuscular dose of 75mg/kg of ceftriaxone sodium (Roche, Nutley, NJ) wasgiven as antimicrobial prophylaxis 30 minutes be-fore the surgical procedures. The gastric pouch wascreated as described in Experiment 2. The biliary-pancreatic limb was lengthened from 16 to 30 cm,and the alimentary limb was established 10 cm belowthe gastrojejunostomy to induce persistent weightloss. The common channel, between the jejunojeju-nostomy and the cecum, was approximately 18 cm.RatsdrankwaterandBoost-Plusstarting24hoursafterthe operation for 7 days, followed by a solid diet. Ratswere hydrated with normal saline solution injectedsubcutaneously as described in Experiment 1. Ratswere euthanized 28 days after the surgical proce-dures. Body weight and operative complications wereevaluated daily.Experiment 4: 20% Gastric Pouch Divided Be-

tweenStaples,Witha30cmBiliary-PancreaticLimb,a 10 cm Alimentary Limb, and a Common ChannelofApproximately18cm. Experiment3wasduplicatedusing 12 obese rats. In 6 a sham operation was per-formed (SO-obese 746.1 � 32.6 g). In 6 obese ratstheRYGB(730.4 � 41.3g)wasdonebyplacinga rowof titanium staple lines (EZ35B, Ethicon, Cincinnati,

OH) between the lesser and greater curvature of thestomach, creating a 20%gastric pouchand transectingthe stomach.The staple linewas placed 2–3mmbelowthe gastro-esophageal junction, preserving the vagusnerve.The rest of theoperative procedure andpostop-erative care was as described in Experiment 3.

Statistical Analysis

Changes inbodyweight,caloric intake,biochemicaldata, and weight of adipose tissue were analyzed usingone-way analysis of variance andmultiple comparisontests by controlling the significance level individuallyas well as jointly at a value equal to P 0.05. When theanalysis of variance test rejected the hypothesis of nodifference, the Tukey’s pairwise multiple comparisonwas applied to ensure that the family (i.e, SO-obese,RYGB, and pair-fed) error rate was equal to 0.05jointly. Data are expressed as mean � standard error.Themodels were considered separately because of thesequential nature of developing our model over aperiod of months. The data from the three modelswere not analyzed jointly or compared to each othersimultaneously.

RESULTSBodyWeight Gain During PreoperativeHigh-Energy Diet or Chow Diet Period

Body weight increased continuously from 54.9 �1.8 g to 492.5 � 6.7 g, as shown in Fig. 2. There wasno difference in body weight gain among the threepreoperative high-energy diet groups. The meanbody weight of the chow-control group was 389.7 �11.7ganddifferedfromthat inrats fed thehigh-energydiet (P � 0.05).

Experiment 1

Operative complications. Of the 24 obese rats thatunderwent operation, 21 had a successful outcome. Intwo rats in the RYGB group, an anastomotic leak oc-curred on postoperative day 3 and 4, whereas one ratin the pair-fed group died during anesthesia. Therewere no complications or deaths in the SO-obesegroup.Postoperative BodyWeight Changes. Body weight

decreased during postoperative days 2 to 4, and thiswas attributed to the effects of anesthesia and the oper-ation. Thereafter bodyweight began to increase in theSO-obese group, which continued on an ad libitumdiet. Body weight increased to 484.4 � 15.4 g by day10, similar to preoperative weight (492.1 � 13.8 g). Inthe RYGB and pair-fed groups, body weight signifi-cantly and continuously decreased after operation.The mean weight loss in the RYGB group was 80 g

Vol. 8, No. 52004 Roux-en-Y Gastric Bypass Model in Diet-Induced Obese Rats 625

Fig. 2. Effect of diet on body weight gain in the diet-induced obese and chow-control groups after 7weeks of a high-energy diet (HED) or chow-control diet, respectively.

(496.9 � 16.3 g to 416.9 � 23.3 g), representing a16.1%weight loss compared to the preoperative con-dition. In the pair-fed group, body weight also de-creased significantly due to food restriction caused bypair feeding. The mean weight loss was 63.7 g (from499.8� 14.9 g to 436.1 � 13.9 g) representing 12.7%of the preoperative body weight. A slight increase inbody weight was noted in three of the six RYGB rats.A contrast study was obtained that showed a gastro-gastric fistula (Fig. 3, A).Caloric Intake. Preoperatively the caloric intake in

theSO-obese,RYGB, andpair-fed groupswasnot sta-tisticallydifferent (129.7 � 5.6 kcal/day, 119.8 � 10.0kcal/day, and 120.7 � 6.2 kcal/day, respectively). Bypostoperative day 10, caloric intake in the RYGBgroup decreased 66% compared to the SO-obese rats(31.4 � 5.8 kcal/day vs. 90.3 � 5.2 kcal/day; P �0.05).

WeightChanges inAdiposeTissues. Fat content inSO-obese rats was higher than that in chow-controlrats (P� 0.05): subcutaneous fat (13.1� 1.2 g vs. 5.8 �0.4 g), retroperitoneal fat (11.5 � 1.0 g vs. 3.9 �0.5 g), mesenteric fat (7.8� 0.8 g vs. 2.5 � 0.2 g), andepididymal fat (8.6� 0.8 g vs. 3.5 � 0.3 g) deposits.IntheRYGBratscomparedtotheSO-obeserats, therewas a significant decrease (P � 0.05) in subcutaneousabdominal fat (7.3 � 0.4 g vs. 13.1 � 1.2 g) and retro-peritoneal fat (8.1 � 1.2 g vs. 11.5 � 1.0 g). Therewasno significant change in mesenteric fat between theRYGBandSO-obese groups (5.8� 1.1 g vs. 7.8 � 0.8g) or in epididymal fat (8.7 � 1.2 g vs. 8.6 � 0.8 g). Inthepair-fedgroupcompared toSO-obesegroup, therewas no significant difference in subcutaneous (11.2 �1.6 g vs.13.1 � 1.2 g), mesenteric (6.9� 1.0 g vs.7.8�0.8 g), retroperitoneal (8.7 � 1.1 g vs. 11.5 � 1.0 g),and epididymal fat (7.7 � 0. 9 g vs. 8.6 � 0.8 g).

Fig. 3. A, Upper gastrointestinal series confirming a gastrogastric fistula. The arrow shows the defunctio-nalized stomach and duodenum containing contrast medium. B, Radiologic study showing no evidencegastrogastric fistula.

Journal ofGastrointestinal Surgery626 Meguid et al.

Biochemical Indices. These are summarized inTable 1. The assumption of no difference in threegroups (SO-obese,RYGB,andpair-fed) isnot rejectedfor free fatty acid concentrations at P � 0.05 by theone-way analysis of variance. For other biochemicalindices, the null hypothesis of no difference is rejectedat P � 0.05; hence Tukey’s pairwise test was con-ducted separately.When comparing pair-fed and SO-obese groups, we found significant decreases based onTukey’s joint tests inplasmaglucose, insulin, triglycer-ide, and leptin concentrations. When comparingRYGB and SO-obese rats, we found that leptin con-centrations were significantly lower in the RYGBgroup based on the joint tests as well. In the case ofSO-obese vs.RYGBrats, inplasmaglucose and insulinconcentrations, significant differences were found inthe individual tests; however, this comparison was notsignificant based on the joint tests.Experiment 2: A higher than expected mortality

rate occurred between postoperative days 1 and 3.Necropsy of the four rats did not reveal major ana-tomic-surgical problems. We attributed these deathsprimarily to the extended anesthesia time and the in-creasedmagnitudeof the intraoperativetrauma.Oper-ative time increased from approximately 50minutes inExperiment 1 to 120 minutes in Experiment 2.Postoperative Changes in Body Weight. The pat-

tern of weight loss in 10 days in this experiment wassimilar to that reported inExperiment1, althoughper-sistent weight loss was not observed. The weight losscurve of the 20% divided gastric pouch, with a 16 cmY limb and a 10 cm alimentary limb, remained belowand ran parallel to the normal weight curve of the con-trol group for the ad libitum diet (Fig. 4, A). An uppergastrointestinal series was performed to rule out thepresence of gastrogastric fistula. This study confirmedtheabsenceofgastrogastricfistulaas showninFig.3,B.The high mortality rate in this experiment sug-

gested that our preoperative, intraoperative, and post-operative management was deficient and not rigorousenough to keep the rats alive for a long period aftersuch amajor surgical procedure. This experiment alsosuggested that the biliary-pancreatic limb should be

lengthened to diminish nutrient absorption so thatthe rats would continue to lose weight.Experiment 3: Fig. 4, B shows weight loss over a

28-day postoperative period. RYGB rats lost 24.3%oftheir original weight (range 541.1 � 6.7 g to 409.4 �10.6 g), and pair-fed rats lost 16.3% of their originalweight (range 546.1 � 7.6 g to 456.8 � 9.3 g). Itshould be noted that the SO-obese rats continued togain weight from day 7 after surgery to the end of theexperiment. The RYGB rats showed sustained weightloss, which was significantly different from the SO-obese and pair-fed rats at the end of the experiment, asshown in Fig. 4,B. In comparison to theRYGBgroup,the weight curve in pair-fed rats showed an initialweight loss but subsequently (from day 14) demon-strated a gradual weight gain.Of the 36 rats studied in this experiment, three rats

in theRYGBgroupdied.All of the rats died of respira-torydistress, suggestingbronchoaspirationonpostop-erative days 1 (two rats) and 4 (one rat). These ratswere replaced to ensure consistent numbers amongthe groups. Of the15 rats that underwent RYGB, 12hadasuccessfuloutcome,constitutinga20%mortalityrate. There were no deaths in the SO-obese and pair-fed groups.Experiment 4: Fig. 5 shows a significant weight

loss over 67 postoperative days. RYGB rats lost 25.4%of their original weight (730.4 � 41.3 g to545.0 � 43.3 g), while SO-obese continued to gainweight from the seventh day after surgery to the endof the experiment (746.1 � 32.6 vs. 788.4 � 33.8 g).Average daily body weight loss per group is summa-rized in Table 2.

DISCUSSION

Obesity, one of the major health problems con-fronting this country, has reached epidemic propor-tion. For the estimated 8 million adults who aremorbidlyobeseandwhoare refractory toconventionalweight loss therapy, a gastric bypass operation is per-formed to reverse the metabolic sequelae of obesity

Table 1. Biochemical indices (mean � standard error)

Group Glucose (mg/dl) Insulin (ng/ml) TG (mg/dl) FFA (mmol/L) Leptin (pg/ml)

SO-obese (n � 8) 173.1 � 8.1 0.85� 0.17 126.1 � 31.5 0.40 � 0.06 944.7 � 147.3RYGB (n � 6) 145.4 � 10.5* 0.46� 0.04* 83.1� 6.7 0.38 � 0.03 452.4 � 133.0†

PF (n � 7) 139.7 � 5.4‡ 0.40� 0.12‡ 51.9 � 5.0†‡ 0.42 � 0.06 338.9 � 61.7†

TG � triglycerides; FFA � free fatty acids.*P � 0.05 vs. SO-obese when analyzed individually.†P � 0.05 vs. SO-obese when analyzed individually and jointly.‡P � 0.05 vs. RYGB when analyzed individually and jointly.

Vol. 8, No. 52004 Roux-en-Y Gastric Bypass Model in Diet-Induced Obese Rats 627

Fig. 4. A, Mean body weight loss at the end of Experiment 2. B, Acute and chronic weight loss inExperiment 3. SO-obese � sham-operated obese; RYGB� Roux-en-Y; PF � pair-fed.

and to induce sustained weight loss. The evolution ofbariatricprocedures thathave led to themost currentlyused operation is based on the following two physio-logic principles: (1) restrict food intake via a small gas-tric pouch and induce satiety; and (2) decrease theamountofnutrients assimilated.TheRYGBoperationencompasses both principles and has thus emerged asthe most common procedure for treating morbidlyobese patients at present. In particular, the ability toperform RYGB via minimally invasive laparoscopicsurgery has made this operation popular.13,20,21 Al-though weight loss certainly occurs, how it occurs andwhy this operation ameliorates obesity-associated dis-eases remains unclear and is not well defined. To fullyunderstand and explore the temporal relationship be-tween weight loss and the amelioration or reversal ofobesity-related metabolic abnormalities, a robust,

well-characterized, and reproducible animal model isneeded. To achieve this goal we are reporting our de-velopmental experience with a RYGB rat model.We initially developed a gastric banding model in

Sprague-Dawley rats by suturing a Silastic bandaround the fundus. No appreciable weight loss oc-curred because the rats continued to eat in an un-restricted manner. At necropsy we found that theSilastic band was intact but the 20%gastric pouch hadballooned out significantly, increasing the food reser-voir. We successfully induced weight loss for 10 daysinobeseZucker ratsusingaRYGBmodel.19Our initialdecision to use Zucker rats was based on the followingtwo factors: (1) our previous extensive experience instudying and publishing studies in the Zucker rats22and (2) in human obesity, a degree of leptin resistanceexists secondary to dietary factors.23 However, this

Journal ofGastrointestinal Surgery628 Meguid et al.

Fig. 5. Body weight after operation in RYGB and SO-obese rats in Experiment 4.

genetic abnormality is a rare and infrequent cause ofobesity in humans. In contrast, diet-induced obesity isthe most common cause of progressive weight gain inindustrialized nations. To study the biochemical andhormonal changes that lead to loss of body weightand amelioration of comorbid conditions afterRYGB,a model of diet-induced obesity in rats was necessary.We induced obesity in Sprague-Dawley rats by

mimicking a typical United States diet consisting ofa high-fat and high-carbohydrate content. Rats feda diet of regular chow served as a control group. Theweight gain in rats on a high-energy diet was signifi-cantly greater compared to chow-fed control rats.After 7weeks, theobese ratswere hyperglycemic, hyp-erinsulinemic, and had enlarged body fat stores. Pre-viously we reported that these obese rats also hadelevated concentrations of corticosteroids and cytok-ines in mesenteric fat depots.16 Having characterizedthe anthropomorphic and metabolic features of theobese rat, we proceeded to perform a series of gastricbypass operations, each building on the experiencegained from the previous experimental model.

In Experiment 1, food intake was restricted via a20% gastric pouch and resulted in a reduction of thefoodintakeby66%(kcal/day)andaweight lossof16%.The subcutaneous abdominal fat and retroperitonealfat in theRYGBratswere significantly decreased com-pared with the SO-obese group. After RYGB, a de-crease in concentrations of glucose and insulin wasmeasured.Thedecreases in triglycerides and in leptin,plus the decrease in the subcutaneous fat mass, reflectthe decreased food intake and the consequentdecreasein body weight, because similar values occurred inthe pair-fed group.Leptin, an anorectic hormonepro-duced by lipocytes, significantly decreased, as ex-pected, because of the decrease in body weight andbody fat content.Thephysical limitationof thegastric pouchmaynot

be the only factor leading to weight loss.24 To test thishypothesis, we included a pair-fed group.When pair-fed rats were provided with the same amount of foodconsumedbyRYGBrats, theweight losswas less com-pared with the RYGB rats. This difference in weightloss between the RYGB and pair-fed groups indicates

Table 2. Mean daily body weight change

Day 0–7 Day 8–30 Day 31–67

Mean daily weight change (gm)SO-obese �8.45 (.71) 1.41 (.12) 1.18 (.06)RYGB �15.44 (1.38) �5.51 (.23) .25 (.11)

Mean initial weight at the beginning of each period (gm)SO-obese 762 690 693RYGB 765 673 504

RYGB � Roux-en-Y.

Vol. 8, No. 52004 Roux-en-Y Gastric Bypass Model in Diet-Induced Obese Rats 629

that the mechanism(s) of weight loss in these twogroups of rats may not be similar. We postulate thatafter RYGB, the small gastric pouch results in early“satiety” signals to thebrain via thevagal afferentnerveand plasma ghrelin, terminating meal size and induc-ing satiation, respectively. In addition, a decrease infood absorption secondary to the 16 cm pancreatic-biliary limb could also contribute toweight loss.How-ever, gastric emptying and small bowel absorptionstudies currently ongoing should shed further lighton the mechanisms of weight loss.We developed an obese rat model that reproduced

the hallmarks of obesity in humans in Experiment 1.FollowingRYGB,weight loss andameliorationofbio-chemical indices of obesity were evident after 10 days.Weight loss beyond 10 days did not occur because ofthe development of suture line fistulas. This led us tomodify the model by dividing the stomach. In Experi-ment 2 wemodified themodel by completely dividingthe stomach to prevent the previous occurrence ofgastrogastric fistula. This procedure significantly ex-tended our operative time. The first rats that under-went RYGB with complete division of the stomachdied during the first 3 days after surgery.Necropsy didnot reveal major anatomic-surgical problems. We at-tributedthesedeathsprimarily to thelongeranesthesiatime and the increased magnitude of the intraoper-ative trauma. However, rats that survived the surgicalprocedure still did not show a sustained weight loss.We performed an upper gastrointestinal series to de-termine the presence of a potential gastrogastric fis-tula. As shown in Fig. 3,B and ultimately via necropsy,no fistulas were demonstrated. The success of thismodification in creating the gastric pouch by gastricdivision became our standard RYGB procedure. Nev-ertheless, persistent weight loss was not observed,motivating us to increase the length of the pancreatic-biliary limb, thereby increasing the length of excludedbowel. However, the most significant contribution ofthis experimentwas relearning the lessonsofperioper-ative care because of the highmortality rate as a resultof this lengthysurgicalprocedure.Thefollowingmod-ifications were performed in our model:

• 20 ml of normal saline solution subcutaneouslypreoperatively to maintain hydration during thesurgical procedure

• A single dose of 75 mg of ceftriaxone as antimi-crobial prophylaxis

• Intraoperative time was shortened from 120 to90 minutes

• 40 ml of normal saline solution subcutaneouslyon completion of every operation

• Heat lamp and hot water bottle tomaintain bodytemperature after surgery

• 20 ml or more of normal saline solution duringthe 3 first postoperative days, depending on thehydration status of the rat

• Postoperative analgesic, buprenorphine hydro-chloride (0.05 mg/kg) twice a day during the first3 days

• Rats were maintained on the liquid Boost-Plusdiet for 7 days instead of 3 days before they werestartedon the coarsely groundchowdiet to facili-tate anastomotic healing

These measures decreased mortality. Althoughtime-consuming, when dealing with many rats simul-taneously, these techniques were adopted as standardoperationalpracticeandwereapplied inExperiment3.In Experiment 3, a 20%divided gastric pouch, with

a 30 cm biliary-pancreatic limb, a 10 cm alimentarylimb, and a common channel of approximately 18 cm,was successfully created. The initial pattern of weightloss was similar to that reported in the two previousexperiments. There was no evidence of a regaining ofbody weight. Instead, a persistent weight loss wasachieved. In contrast, it should be noted that the SO-obese rats continued to gain weight. This is a commonphenomenon in rats that tend to slowly gain weightthroughout their lifespan. In comparison to theRYGBgroup, the weight curve in pair-fed rats showed an ini-tialweight loss but subsequently (fromday10) demon-stratedagradualweightgain.Thisfindingsuggeststhatin the presence of the lengthened intestinal bypass,diminishednutrient absorption is a factor contributingto continued andpersistentweight loss inRYGB.Thisclinical impression is supported by our observationsthat the rats’ feceswere less formed andwetter, as indi-cated by the wider staining surrounding the feces onthe pan liner under each cage. Pair-fed rats gainedweight 10 days after operation, whereas RYGB ratsshowed persistent weight loss. This suggests that themechanism(s) of weight loss in the latter is certainlydifferent from that in pair-fed rats, which calls for fur-ther studies. Now that we have developed a reproduc-ible model of RYGB such studies are in progress.However, the value of a sustained weight loss for

28 days was initially questioned in our studies, and amortality rate of 20% needed to be improved. Hencewe embarked on Experiment 4, in which we used anEZ35B stapler to create a 20% gastric pouch totransect the stomach. This decreased the operativetrauma of our previousmodel, in which the pouchwascreated manually. Our operative time decreased, andthe rats awakened faster and were more active duringthe first postoperative days. Finally, this modificationalso decreased our mortality rate.Theexistenceofa robust, reliable, and reproducible

rat model of RYGB opens a new field in the research

Journal ofGastrointestinal Surgery630 Meguid et al.

of obesity. This reliable RYGB permits us to investi-gate themechanismsofweight loss afterRYGB,aswellas the mechanisms of improvement of the diseases re-lated to obesity, including diabetes, heart disease andhypertension, and wound healing.

WethankBoost-PlusMeadJohnson (Evansville, Indiana) for supply-ing the Boost-Plus used in this study.

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