To investigate the lymph network change in pancreatic duct obstruction in pigs as a model of pancreatic cancer invading pancreatic duct, six domestic pigs weighing 17-40 kg underwent surgery as protocol. Two of them were controls, and the others underwent ligation of the pancreatic duct as a model of ductal obstruction. A CH 40 and lipiodol mixture was injected in their pancreas at 7 or 21 Days after first operation. Radiographic examination had been also performed. Five or 14 days later, they were examined radiographically, and sacrificed for histological examination. Ligation of the pancreatic duct caused experimental pancreatitis. Dilatation of the pancreatic duct and dilatation of the lymph canal in the interlobular space of pancreas was demonstrated in the ligation group. CH 40 and lipiodol showed discrepancies in the distribution. There were not distinct differences between the two groups in a route of CH 40 traveling. Only fluoroscopic examination revealed an image of lipiodol enlargement to the caudal site in the ligated group. The congestive lymph system may have impaired flow like reflux

Kurahashi, Miho

Miyake, Hidenori

Takagi, Toshihide

Tashiro, Seiki

Tokushima University Institutional Repository

INTRODUCTIONPancreatic cancer has been themost lethal diseaseof whole alimentary tract carcinomas in Japan (1). Thisis due to the high frequency of recurrence in the retrop-eritoneal space even after radical resection for pan-creatic carcinoma. Cancer cells easily spread to theretroperitoneal space even in case of early stage ofpancreatic carcinoma. Metastasis has often occurredin the liver, and the paraaortic lymph nodes.The reason for the high frequency of recurrencein the retroperitoneal space is that residual cancer cellsremain in the pancreatic bed. These are not detectedmicroscopically at the time of operation. Also the tumorcells grow in to recurrence lesions in the retroperitonealspace, we have often found tumors to be widespreadthere.It has been speculated that the pancreatic paren-chyma connects to the retroperitoneal space with therich network of lymph vessels. Sometimes it has beenshown that tumors of the pancreatic head invade thepancreatic duct and cause obstruction of the pancreaticduct.We made a model of pancreatic duct obstruction inthe pigs, and evaluated the lymphsystemchangesunderthe special condition like pancreatic duct obstruction.MATERIALS AND METHODSAnimals : Seven male pigs (19-40 kg body weight)were prepared for experimental animals. After all,six male pigs were used as experimental animalsexcept one had died of intestinal obstruction. TheyORIGINALChanges of lymphatic flow incaseofpancreaticductobstructionin the pig -as a model of pancreatic cancer-Miho Kurahashi, Hidenori Miyake, Toshihide Takagi, and Seiki TashiroDepartment of Digestive and Pediatric Surgery, The University of Tokushima School of Medicine,Tokushima, JapanAbstract : To investigate the lymph network change in pancreatic duct obstruction in pigsas a model of pancreatic cancer invading pancreatic duct, six domestic pigs weighing17-40 kg underwent surgery as protocol. Two of them were controls, and the others underwentligation of the pancreatic duct as a model of ductal obstruction. A CH 40 and lipiodol mixturewas injected in their pancreas at 7 or 21 Days after first operation. Radiographic examinationhad been also performed. Fiveor14dayslater,theywereexaminedradiographically,andsacrificedfor histological examination. Ligation of the pancreatic duct caused experimental pancreatitis.Dilatation of the pancreatic duct and dilatation of the lymph canal in the interlobularspace of pancreas was demonstratedin the ligationgroup.CH40andlipiodolshoweddiscrepanciesin the distribution. There were not distinct differences between the two groups in a routeof CH 40 traveling.Only fluoroscopic examination revealed an image of lipiodol enlargement to the caudalsite in the ligated group. The congestive lymph system may have impaired flow like reflux.J. Med. Invest. 51 : 70-75, February, 2004Keywords : lymph system, pig, obstruction, pancreatic duct, pancreatic cancerReceived for publication December 1, 2003 ; acceptedDecember 25,2003.Address correspondence and reprint requests toMiho Kurahashi,M.D., Department of Digestive and Pediatric Surgery, TheUniversityof Tokushima School of Medicine, Kuramoto-cho, Tokushima,770-8503, Japan and Fax : +81-88-631-9698.The Journal of Medical Investigation Vol. 51 2004７０were all AWAPORK, and fed nothing orally exceptwater the day before operation.Anesthesia . Anesthesia was induced with ketaminehydrochloride (10mg/kg) and atropin sulfate (0.01～0.02 mg/kg) with intramuscular injection. The sedatedpig was given bromo vecronium as a muscle relaxantand thiamilal intravenously. After positioning of en-dotracheal tube, a ventilator was connected to the tubefor delivering 35% oxygen. Anesthesia was maintainedwith a low dose of bromo vecronium and pentobarbitalintravenously.Operation. Six pigs underwent laparotomy. Threeof the six pigs underwent a second laparotomy insteadof sacrifice. The pigs received an upper midline skinincision following a right subcostal skin incision .The model of pancreatic duct obstructionwasmadeaccording to the method of Waterworth et al. (2). Thepancreatic duct entered the second part of duodenumat the end point of the pancreatoduodenal attachment(3). The common bile duct was independent of thepancreatic duct, it entered the upper duodenumnearthe pyloric ring. Duodenotomy was performed at theopposite site of the pancreatic duct orifice. The orificewas exposed and a purse string suture was performedto ligate the pancreatic duct two or three times with5-0 non-absorbable monofilament nylon. Duodenotomywas repaired with a longitudinal incision to the hori-zontal suture, without making a narrow lumen of theduodenum. The peritoneum and abdominal wall wererepaired with absorbable thread. Skin was fixed witha metal stapler. The surgical woundwas covered withsurgical skin aerosol. All the procedureswere performedunder aseptic conditions. After the operation, the pigswere fed nothing orally for 12 hours. Drip infusionwascontinued via the ear vein until it was spontaneouslydisconnected. (When the pigs stood up and walkedaround, the infusion line was disconnected.) Optimumtemperature was maintained on the pig floor in theanimal laboratory of University of Tokushima, especiallyafter the operation. Antibiotic agents and H2-blockerwere injected on the operative day only.LIPIODOL. Lipiodol Ultra-Fluid is ethylester ofiodinated poppy-seed oil fatty acids. It is used for lym-phangiography in clinical conditions. Lipiodol and CH40 were mixed just before injection, and it was injectedmanually to the pancreatic body at a rate of 0.5ml perminute.CH40. CH40 is activated carbon particles. It is takenup selectively by the lymphatic system and regionallymph nodes. The route of CH40 travel was stainedblack. The lymph nodes containing CH40 were black-ened.Experimental ProtocolGroup 1. No obstruction of pancreatic ductTwo of the six pigs which did not have pancreaticduct obstruction were injected with the agent at theinitial operation (Table 1). They didnotundergo surgeryof ligation of the pancreatic duct, andwere designatedno-ligation group. Case A was underwent injectionwith lipiodol only. The lipiodol was injected at pancreaticbody just below the pancreatic capsule. Case A wasreceived radiography at Day 7, 25, and 205. It was sac-rificed, and the organswere taken. An other pig, CaseB also underwent injection with CH40 and lipiodol atthe same site of case A. Case B received radiographyone and two weeks after the operation, was sacrificed,and the organs were taken.Group 2. Pancreatic duct obstructionFour pigs, Case C,D, E, F underwent surgery asmodels of pancreatic duct obstruction. Case C wassacrificed at Day 7 after ligation for the purpose ofexamination whether pancreatic duct dilated or notunder the pancreatic duct ligation. Case D was per-formed ligation, and seven days after, been injectedwith a mixture of CH40 and lipiodol. Case E and Fwereunderwent a second surgery threeweeks after the firstoperation. At that time, they were injectedwith amix-ture of CH 40 and lipiodol and to the pancreatic pa-Table 1. Study protocol of six pigsPigpancreaticductduration fromligation to injectioninjection agent (ml)CH 40 lipiodolinjection site inthe pancreasduration from injectionto sacrificeA no ligation － － 2.5 body 7 monthB no ligation － 1.34 2.67 body 14 daysC ligation － － － － 7 daysD ligation 7 days 0.5 1.0 body 14 daysE ligation 21 days 0.66 1.33 body 7 daysF ligation 21 days 0.67 1.33 body 5 daysThe Journal of Medical Investigation Vol. 51 February 2004 ７１renchyma. They were sacrificed at the almost sev-enth day, and consecutive extirpation was performedto take the abdominal organs.EXTIRPATIONANDHISTOLOGICALEXAMINATIONAt necropsy, for the examination of the pancreaticlymph network, not only the pancreas but also thenearby organs and additional tissue were extirpateden masse. The upper margin was the caval vein at thesubphrenic level. The oral surgical margin was at theupper duodenum just below the common bile ductentrance. The portal vein was ligated at the same level.The bilateral kidney, adrenal glands and spleen wereincluded in the resected block, and the anal marginwas the small intestine at the level of the commoniliac vein. The lowermarginwas the common iliac vein.The resected blockwas fixed in 10% formalin, embededin paraffin, and stained with hematoxylin and eosin.RADIOGRAPHIC EXAMINATIONFor the tracing of Lipiodol, the pigs underwent ra-diographic examination usingketaminehydroxychlorideand atropin sulfate.RESULTSGroup 1. No obstruction of the pancreatic ductCase A was from the control group, and underwentaseptic laparotomy. The pig received the injectionof the lipiodol only in the pancreas. At the seventhday after operation, the sedated pig was examinedradiographically. The opacity in the abdomen wasdue to the lipiodol. Radiographic examination wasagain performed at the eighteenth day after opera-tion. The shadow of the abdomen changed in shapebut had not moved very far. It seemed that the changeof shape was only due to a difference in intestinalfullness. The next radiographic examinationwas per-formed six months later. The opacity of the lipiodolhad not moved but had become hazy.Case B received the injection of the lipiodol-CH40mixture in the pancreas. The seven days after operation,the pig was examined radiographically. The opacityin the abdomen had spread. However, the opacity hadnot moved to farther lymph nodes. Another sevendays later, another exploration was performed. Theradiography revealed that the shape of the shadowhad enlarged slightly. After sacrifice, histological find-ings revealed that the CH40 had flowed to the paraaorticadjacent tissue at the level of the phrenic muscle andthe pancreas top.However, theparaaortic lymphsystemdid not contain CH40.There was no CH40 in the caudalparaaortic lymph nodes. The most caudal slice withCH 40 was at the pancreas end and regional fatty tissue.The slice consisted of adrenal gland, kidney, phrenicmuscle, and pancreas. A particle of lipiodol was detectedin the peripancreatic fatty tissue. It had not moved toany lymph nodes histologically (Table 2).Group 2. Pancreatic duct obstructionAll pigs with pancreatic duct obstruction developedmicroscopic changes of acute pancreatitis at the sec-ond operation. The characteristic findings were“acinercell homogenization”pathologically. The pancreaticduct was dilated, and the parenchyma became atro-phic, and showed inflammatory reactions, with plasmacells, lymphocytes and eosinophils. Infiltration ofplasma cells, lymphocytes and eosinophils was shownas a mixture in the acini,“homogenization”. Fibrosisand proliferation of the interlobular connective tissuewas also shown. Hemorrhage was slightly detectedin the fatty tissue. The perilobular space was changededematously.Ligation of the pancreatic duct obviously causedexperimental pancreatitis, and it was also consideredthe obstruction due to pancreatic tumor invasion. CaseC underwent ligation of pancreatic duct, showed dila-Table 2. The distribution of the CH 40Lymph node Connective tissuepigperipancreatic paraaortic peripancreatic interlobular paraaorticsupra infra supra infra supra infraB ＋ ＋ － － ＋ ＋ ＋ －D － － － － ＋ － － －E ＋ － － ＋ ＋ ＋ ＋ －F ＋ － － － ＋ ＋ － －M. Kurahashi et al. Lymphatic flow in case of pancreatic cancer７２tation of pancreatic ducts, fibrosis, and inflammaticchanges histologically.Case D received the injection at the seventh dayafter the first operation of ligation of the pancreaticduct. The pancreas was reddish, but not seriously.The pig was sacrificed two weeks after the secondoperation. Histological findings revealed that CH40existed only in the pancreatic connective tissue. Thelymph system showed dilatation in the pancreas. CH40 was not found in the peripancreatic lymph nodenor the paraaortic lymph nodes. However, coloredlymph nodes were macroscopically distinct, the picricacid process revealed the colored region with a for-malin stain not CH40. Radiographic examination ofthe pig revealed the opacity in the pancreas. The opacitywas due to accumulation of lipiodol, though histologicalfindings failed to detect the lipiodol. The radiographicexamination after the second operation demonstratedthat the lipiodol had flowed into the dilated pancreaticduct. As the experimental pancreatitis was not severe,the lipiodol may have moved to the duodenal lumenby the papilla passage. We speculated that the CH40was also flushed out during the two weeks after theoperation.Case E and F received the injection at three weeksafter the first operation. At the second operation, thepancreas showed redness but not seriously. Aftersuccessful injection, Case E was treated as the otherpigs and sacrificed at the7days after the operation.The radiographic examination revealed that the lipiodolshadow of Case E was not changed. However, CaseF, which was sacrificed at the 5 days after the secondoperation revealed that the lipiodol shadow was thelargest in the series (Fig.1, 2). The shape of lipiodolhad progressed toward even caudal site during threeweeks. It was contrary to customed expectations.Histological findings of Case F revealed that CH40had spread intomany lymph nodes and the interlobularspace of the pancreas. The most cranial lymph nodewhich containedCH40was at the level of the pancreastop. There was phrenicmuscle and abdominal aorta inthe same slice. The CH40was found in the interlobularspace of the pancreas, and in the dilated lymph canalin the interlobular space. Themost caudal lymph nodewith CH40 was at the level of the kidney, adrenal gland,and pancreas end. Lipiodol was not found in any speci-men histologically. Case E had foundCH40 in theperi-pancreatic fatty tissue, peripancreatic lymph nodes,and interlobular space of the pancreas histologically.a bFigure 1. The fluoroscopy showed the lipiodol image at the time of injection to case G.The Journal of Medical Investigation Vol. 51 February 2004 ７３DISCUSSIONThere have been no studies of the lymph systemof the pancreas under pancreatic duct obstruction.We made a model of pancreatic duct obstruction asa model of pancreatic cancer invasion. Clinically, weexperienced that the contrast medium injected intothe pancreas spread to the retroperitoneal spacewidely.One case had advanced cancer of the pancreatic headwith obstruction of the pancreatic duct, with progressof recurrence to the retroperitoneal space thereafter.Therefore, a hypothesis of the existence of a lymphnetwork between the retroperitoneal space and thepancreas in man has been built.We speculated that theobstruction of pancreatic duct caused lymph systemchange, cancer cells might have spread to unexpectedregion. Our model of pancreatic duct obstruction as-sumed that the cancer of the pancreatic head had in-vaded the pancreatic duct. Pancreatic cancer commonlyoriginates from the epidermal layer in the pancreaticduct, so obstructed the pancreatic duct. In general,a lymph flow has been gathered into lymph node andgone to more proximal lymph system. The most im-portant fact is that a lymph flow always goes towardproximately. Only Case F had shown a change oflymph system in the study with lipiodol. The shapeof lipiodol of Case F enlarged not only upward but alsodownward, to caudal site. However, the results of dis-tribution of the CH40 was not unexpected. The CH40was injected in the subcapsular space of the pancreas,flowed into a small lymph canal of interlobular spaceof the pancreas, moved to regional lymph nodes andreached to more proximal lymph nodes. It seemedthat CH40 travel an ordinary route. Even if somewereinjected in peripancreatic connective tissue, they trav-eled in the loose tissue like paraaortic connective tissuein the pigs.The discrepancy of the CH40 distribution and lipiodollocation was detected in the all of the pigs. The reasona bFigure 2. The fluoroscopy showed the enlargement of the lipiodol image 5days after injection in the Case G. The imagewas enlarged upward and downward.Figure 3. The CH40 was detected in the peripancreatic tissue,interlobular space, and lymph canal within the interlobular space.(H & E×200)M. Kurahashi et al. Lymphatic flow in case of pancreatic cancer７４for this was that CH40 had tendency tomove to fartherlymph nodes. In contrast, the lipiodol ultrafluid hadnested in focus, as it had an affinity to oil. In this study,the histological examination could not detect lipiodolexcept in Case B.Macrophageswith pigment granuleswere detected in some slices histologically. However,there was no evidence that the granules originatedfrom lipiodol. The lipiodol ultrafluid was dissolovedby the ethanol, benzene and ether. The process of tissuestaining could have effected the results.Morever, thelipiodol ultrafluid was produced for the purpose oflymphangiography. If it has not been injected into thelymph system, the lipiodol remains a lipids compoundand is stored (4). Dogswhich had received an injectionof lipiodol to their lymph system demonstrated persis-tent images in their lymph nodes for one or twoweeksradiographically (5). The lipiodolmay not be a properagent for the pancreas in this series. We can not tellwhether the lipiodol succeeded in reaching the lymphcanal in the pancreas, because the diameter of the par-ticle is not known. Only fluoroscopic exploration canshow the enlargement of the lipiodol image movingin a caudal site object to ordinary flow. This showsthe feasibility of lymph system change after pancre-atic duct ligation. The ligation can produce dilatationof the panceatic duct, and the high ductal pressuremay affect the connective tissue in the interlobularspace of the pancreas. The congestive lymph systemmay have impaired flow, like reflux. The duration ofcongestion did not show a difference in distributionof CH40. There are anatomical differences betweenpigs and man : the pancreas of the pig lies in the intes-tinal mesenterium, free from the retroperitoneum. Theorifice of the pancreatic duct is independent from thebiliary system. The abdominal aorta is covered inmus-cle bundles which are concomitant to the diaphragm.The aorta and muscle bundles are separate, only con-nected with thin membranous connective tissue. Nev-erthless we can refer much anatomical differencesman and pigs, the impaired flow like reflux in Case Fmight have occurred in man under the same condi-tion, like pancreatic duct obstruction due to cancerinvasion.REFERENCES1. Miyazaki I, Nakagawa T : Approach to pancreas-tic Cancer. Kanehara & Co.,Ltd.,Tokyo, Japan,1997, pp 5-182. Waterworth MW, Barbezart GO, Hickman R,Terblanche J : A controlled trial of glucagonsin experimental pancreastitis. Br J Surg 63 :617-620, 19763. Schroder T, Ramo OJ, Joffe SN : Laser Pncre-atectomy, a comparison between Dog and Pig.Res Exp Med 188 : 227-233, 19884. Viamote M, SotoM, Recher L : Chronic toxicityStudy in dogs Following Intralymphatic injec-tions of ethiodol, ethiodol with Chlorophyl andSterlie Non-Iodnized Poppy Seed Oil. Ind MedSurg３５: 688-695, 19665. Schaffer B, Koehler PR, Daniel CR, Wohl GT,Rivera E, Meyers WA, Skelley JF :A critical-evaluation of Lymphangiography. Radiology 80 :917-930, 1963The Journal of Medical Investigation Vol. 51 February 2004 ７５

Changes of lymphatic flow in case of pancreatic duct obstruction in the pig : as a model of pancreatic cancer

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Changes of lymphatic flow in case of pancreatic duct obstruction in the pig : as a model of pancreatic cancer

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