Journal of Investigative Surgery, 13:45± 57, 2000

Copyright ° c 2000 Taylor & Francis0894-1939/00 $12.00 + .00

New Methodologies

A Canine Model of MultiplePortosystemic Shunting

Lisa M. Howe, DVM, PhD,

Harry W. Boothe, Jr., DVM, MS,

and Matthew W. Miller, DVM, MS

Department of Small AnimalMedicine and Surgery,Texas Veterinary Medical Center,Texas A&M University,College Station, Texas, USA

Dawn M. Boothe, DVM, PhD

Department of Veterinary Physiologyand Pharmacology,Texas Veterinary Medical Center,Texas A&M University,College Station, Texas, USA

ABSTRACT The objective of this study was to develop and describe anexperimental canine model of multiple acquired portosystemic shunts(PSS) similar in nature to spontaneously occurring PSS. Sixteen dogs wereused and were divided into a control (n = 6) and a diseased group (n = 10).Dogs of the diseased group were administered dimethylnitrosamine(2 mg/kg of body weight, po) twice weekly, and clinicopathologic, ul-trasonographic, and hepatic scintigraphic � ndings were recorded duringthe development of hepatic disease and PSS. Surgery was then performedto permit visual veri� cation of multiple shunts, catheter placement forportography examination, and biopsy of the liver. All diseased dogs de-veloped severe hepatic disease and multiple PSS as documented visuallyat surgery and on portography. Based on this study, dimethylnitrosamine-induced portosystemic shunting appears to be an appropriate model forspontaneously occurring multiple PSS secondary to portal hypertension.

KEYWORDS canine, dimethylnitrosamine, disease model, hepatic cirrhosis, hep-atic disease, portal hypertension, portosystemic shunting

Dimethylnitrosamine (N -nitrosodimethylamine) is a hepatic-speci� c toxin that consistently produces hepatic � brosis and cir-rhosis in dogs following chronic administration [1, 2]. Dimethyl-

nitrosamine (DMNA) is an N -alkyl-N -nitroso compound that has thefollowing structure [3]:

The nitrosamine compounds, and especially DMNA, have been stud-ied since the 1950s in an effort to learn about their potential toxicities[3–13]. Several species of animals, including dogs, have been used as

Received 17 November 1998;accepted 9 June 1999.

Supported by a grant from the Doris andHarry Starr Fund, Development Foundation,

Texas A&M University.

Address reprint requests to Lisa M. Howe,Department of Small Animal Medicineand Surgery, Texas Veterinary Medical

Center, Texas A&M University, CollegeStation, TX 77843-4474, USA.

45

New Methodologies

models for DMNA-induced toxicity [1, 2, 9]. Thetwo major toxicities of DMNA are centrilobular hep-atic necrosis with � brosis (cirrhosis), and carcinog-enicity [1, 2]. Intermittent administration of DMNAproduces progressive hepatic cirrhosis, with its sever-ity being dose and time dependent [1, 2]. Multi-ple portosystemic shunts (PSS) have been noted atnecropsy in dogs with experimentally induced cir-rhotic hepatic disease [1, 2].

Multiple portosystemic shunts in both humansand animals develop and become functional whenhepatic damage is suf� cient to increase resistance toportal blood � ow for a suf� cient period of time. In-creased resistance of suf� cient duration accompaniesthe structural changes in hepatic architecture as thedisease progresses and results in chronic portal hy-pertension with subsequent multiple PSS. Since thetoxicity of DMNA is speci� c for the liver, it shouldproduce hepatic changes that result in increased re-sistance, portal hypertension, and acquired PSS.

The objective of this study was to develop and de-scribe a model of canine multiple PSS secondary toDMNA-induced hepatic disease useful to both hu-man and veterinary scientists researching portal hy-pertension and/or portosystemic shunting. The re-sults reported here represent a portion of a muchlarger study [14, 15].

MATERIALS AND METHODS

Animals and Animal Care

Sixteen young adult, medium-sized (26 § 6 kg),mixed-breed dogs (8 males, 8 females) were assignedrandomly to either a control (Group 1, n = 6) ora diseased (Group 2, n = 10) group. Animals wereacclimated to the hospital environment, vaccinated(Imrab, Pitman-Moore, Inc., Mundelein, IL and Van-guard 5/L, Norden Laboratories, Inc., Lincoln, NE),and dewormed (Panacur, Hoechst-Roussel, Agri-VetCompany, Somerville, NJ) at least 6 weeks before thestudy. Dogs were housed individually at the teachinghospital and were fed a standard hospital diet unlessascites developed, which necessitated dietary man-agement. The experimental protocol was reviewedand approved by the University Laboratory AnimalUse and Care Committee prior to initiation of the

study. Animals were cared for and the study was con-ducted in a manner consistent with the U.S. NationalInstitutes of Health Guide for the Care and Use ofLaboratory Animals and the Animal Welfare Acts(US PL 89-544; 91-579; 94-279).

Prestudy Evaluation

All dogs were subjected to a prestudy evaluation todetect any illnesses prior to initiation of the study.Routine physical examinations were performed oneach dog. Clinical laboratory testing was also per-formed and consisted of those tests to be performedthroughout the study: complete blood count (CBC),platelet count (PLAT), serum chemistry pro� le con-sisting of albumin (ALB), alkaline phosphatase(ALKP), alanine aminotransferase (ALT), total biliru-bin (BILI), blood urea nitrogen (BUN), cholesterol(CHOL), glucose (GLU), gamma-glutamyltransfe-rase (GGT), and total protein (TP). Additional clin-ical laboratory tests performed included a coagu-lation pro� le consisting of an antithrombin IIIactivity (AT3), activated prothrombin time (PT), andpartial thromboplastin time (PTT), a pre- and 30-min postprandial ammonia tolerance test (PRNH3and PONH3), a pre- and 2-h postprandial bile acidconcentration (PRBA and POBA), a 30-min sulfo-bromophthalein (BSP) retention time, and a 45-minindocyanine green (ICG) clearance study. Lastly, rou-tine urinalysis, fecal examination, heartworm test,and Ehrlichia test were performed, as were abdom-inal radiographs and ultrasonography, and hepaticscintigraphy. All tests were normal before continu-ing with the study.

The sequence of sample collection was as follows.With the exception of postprandial tests, all bloodsamples were collected from fasted dogs. Blood forthe CBC, serum chemistries, coagulation pro� les,and fasting ammonia and bile acids was collected.Sulfobromophthalein was then injected intravenou-sly (5 mg/kg). The 30-min BSP blood sample wascollected and the animals were then fed one can ofdog food (Canine P/D, Hill’s Pet Nutrition, Inc.,Topeka, KS). Thirty minutes later, the postprandialammonia sample was taken, and then the 2-h post-prandial bile acid blood sample was drawn. Bloodammonia samples were placed immediately in ice

46 L. M. HOWE ET AL.

New Methodologies

after collection and taken to the laboratory within15 min of collection. Other blood samples weretaken to the laboratory within 60 min of collection.

Indocyanine green (ICG; Cardiogreen, FlukaChemical Corporation, Rnokonkoma, NY) serumclearance studies were performed on a different daythan other clinical laboratory testing using previ-ously described methods [16]. Brie� y, indocyaninegreen studies were conducted immediately follow-ing preparation and � ltration of an ICG solution(10 mg/ml) using a 0.45- l m millipore � lter (Mil-lipore, Millipore Corporation, Bedford, MA) intoa sterile injection vial. Indwelling cephalic catheterswere placed aseptically for blood withdrawals. Clear-ance studies were performed by injecting ICG(0.5 mg/kg iv using the cephalic vein not containingthe catheter) following collection of a 0 time sam-ple and collecting blood samples at 3, 6, 9, 12, 15,20, 30, and 45 min. An ultraviolet spectrophotome-ter (Beckman spectrophotometer, Beckman Instru-ments, Inc., Fullerton, CA; wavelength of 805 nm)was used to determine ICG concentration within24 h of collection [16].

Abdominal ultrasonographic examinations [17]were focused primarily on the cranial abdomen (liverand region of the kidneys). Hepatic scintigraphy wasperformed using intravenously injected technetium99m-sulfur colloid (AN-Sulfur Colloid, CIS-US,Inc., Bedford, MA) according to previously estab-lished guidelines [18–20].

Dimethylnitrosamine Preparation

Dimethylnitrosamine capsules were prepared andstored in strict adherence to the Occupational Safetyand Health Administration guidelines for thehandling of toxic materials. Dimethylnitrosamine(N -nitrosodimethylamine, Sigma Chemical Co.,St. Louis, MO) was administered (2 mg/kg po twiceweekly on consecutive days) in gelatin capsules con-taining dextrose as a vehicle to dogs in the studygroup until multiple PSS were presumptively doc-umented. The dose of DMNA [1, 2] was adjustedweekly for individual response (based on clinicallaboratory and hepatic clearance test results) toprevent clinical manifestations of severe hepaticdisease.

Monitoring Hepatic Disease andDevelopment of Multiple PSS

Dogs were observed daily, and physical examina-tions were performed every other day. Routine clin-ical laboratory testing and tests of hepatic functionwere repeated every 14 days as previously describeduntil surgery. Ultrasonographic and hepatic scinti-graphic examinations were repeated every 21 daysuntil surgery. Final clinical laboratory testing, ultra-sonographic, and hepatic scintigraphic examinationswere performed within 1 week of surgery in all dis-eased animals.

Determination of Endpoint ofModel Development

Multiple PSS were considered present when phys-ical examination � ndings, clinical laboratory tests,and tests of hepatic function were consistent withhepatic disease and multiple portosystemic shunt-ing. Clinical laboratory tests used to determine pro-gression of hepatic disease and suspected presence ofmultiple PSS included tests that were indicative ofhepatic damage (ALT), cholestatic disease (ALKPandGGT), reduced hepatic clearance (PRNH3, PONH3,PRBA, POBA, BSP, and ICG), and reduced hepaticprotein synthesis (ALB, TP, PT, PTT, and AT3) [1].Other miscellaneous tests included CHOL, BUN,and BILI. Additionally, multiple PSS were consid-ered to be present when suspicious vessels were notedon abdominal ultrasonography in the region of theleft kidney, and characteristic hepatic scintigraphic� ndings (elevated hepatic perfusion indices) werenoted [17–22]. Dimethylnitrosamine administrationwas discontinued once PSS were presumptively doc-umented, and surgery was performed the followingweek.

Anesthesia and SurgicalPreparation

All dogs underwent the same anesthetic and sur-gical procedure. Dogs were premedicated with gly-copyrrolate (Robinul-V, Fort Dodge Laboratories,Fort Dodge, IA), and anesthesia was induced usingiso� urane (Aerrane, Anaquest,Madison, WI) admin-istered via a mask. Anesthesia was maintained with

A MODEL OF MULTIPLE PORTOSYSTEMIC SHUNTING 47

New Methodologies

iso� urane in oxygen delivered by a semiclosed anes-thetic system through a cuffed endotracheal tube.Dogs were prepared for aseptic abdominal surgery.

Surgical Placement of VenousCatheters

After a ventral midline abdominal incision be-ginning at the level of the xiphoid process and ex-tending to the umbilicus, the presence (Group 2) orabsence (Group 1) of multiple PSS was veri� ed. Anindwelling silastic catheter (Silastic medical gradetubing,DowCorning Product,Midland,MI; 0.10 cmID) was placed in the portal vein at the hilus of theleft lateral hepatic lobe and secured to the vessel with3-0 silk suture (Silk Suture, Davis & Geck, Ameri-can Cyanamid Co., Danbury, CT). The tip of thecatheter was directed caudally, and its location levelwith the gastrosplenic vein was veri� ed digitally.

An hepatic biopsy was obtained from the left me-dial lobe, and the abdomen was closed, leaving thefree end of the portal catheter exposed. Contrast por-tography was performed (6 radiographs over 3 s) dur-ing intravenous injection of 0.25 ml/kg contrast ma-terial (Renogra� n, Solvay Veterinary Inc., Princeton,NJ) into the portal catheter. Following completionof the contrast study, the animal was again preparedfor and returned to surgery for additional studies[14, 15].

Pharmacokinetic Analysis

A standard pharmacokinetic analysis was per-formed for ICG after the determination of drug con-centrations in all samples. Serum drug concentrationversus time curves were analyzed initially using aleast-squares nonlinear regression computerprogram(R-Strip) to estimate initial pharmacokinetic param-eters (R-Strip, Micro Math, Inc., Salt Lake City, UT).Final parameter estimates were made using a nonlin-ear least-squares regression program (PCNONLIN,Statistical Consultants, Inc., Lexington, KY). Indo-cyanine green parameters studied and compared in-cluded area under the curve (AUC), area under themoment curve (AUMC), clearance (CL), maximumconcentration (CMAX), overall elimination rate con-stant (K10), elimination half-life (K10HL), mean res-

idence time (MRT), and apparent volume of distri-bution (V d).

Statistical Analysis

Comparisons for clinical pathology testing resultsand hepatic perfusion indices between groups andamong times within groups were made using a gen-eral linear model with repeated measures (SAS Sys-tem, SAS Institute, Inc., Cary, NC). Duncan’s mul-tiple range testing was used to identify signi� cantdifferences. Differences were considered signi� cantat p · .05.

RESULTS

Clinical Signs and LaboratoryTesting

Orally administered DMNA consistently pro-duced multiple PSS in all diseased animals, as sug-gested by ultrasound and hepatic scintigraphy, anddocumented at celiotomy and operative portography.Based on physical examination � ndings, alterationsin clinical laboratory tests, and ultrasonographic andhepatic scintigraphic � ndings characteristic of mul-tiple PSS, 8 of 10 dogs developed shunts within8–14 weeks, while 2 dogs required more prolongedDMNA administration (40 and 56 weeks). The lengthof time of DMNA administration to each dog in thediseased group is listed in Table 1.

Dimethylnitrosamine administration producedmild clinical signs indicative of hepatic disease at

TABLE 1 Duration (weeks)ofdimethylnitrosamine(DMNA)administration to Group 2 dogs

Weeks of DMNADog administration

052 7059 8159 9250 7296 56300 6301 10303 8308 6335 40Mean § SD 15.7 § 17.5Median 8

48 L. M. HOWE ET AL.

New Methodologies

TABLE 2 Temporal onset (weeks) of clinical signs inGroup 2 dogs

Clinical sign (weekN of ® rst observation) Mean § SD Median

9 Fever (>104±F) 6.9 § 4.87 7.09 Anorexia 7.2 § 2.9 6.09 Ascites 17.4 § 18.1 8.06 Weight loss 14.3 § 14.6 8.52 Icterus 9.0 § 4.2 9.0Note. SD, standard deviation.

the same time test results (including abdominal ul-trasound and hepatic scintigraphy) were becominghighly suggestive of PSS (usually 2–3 weeks priorto surgery). Temporal onset of clinical signs in thediseased group is listed in Table 2. Clinical signsincluded fever (n = 9), ascites (increased abdomi-nal girth measurement and ballotment of � uid inabdomen; n = 9), intermittent anorexia (n = 9),weight loss (2–15% body weight; n = 6), and icterus(n = 2). Clinical signs became more obvious withprogression of hepatic disease.

The temporal changes seen in clinical pathologytest results are listed in Table 3. The most severeabnormalities were seen during weeks 8–12 when8 of the dogs’ laboratory test results indicated hep-atic disease and probable PSS. Clinical laboratorytests in the diseased group, which differed signi� -cantly from both the control group and baseline val-ues and continued to change with the progressionof hepatic disease, included ALT (p < .01), ALKP(p < .0001), PCV (p < .0003), PLAT (p < .001),PRNH3 (p < .0003), PONH3, PRBA, POBA, BSP,ALB, PTT, AT3, BUN (all: p < .0001), and ICG(discussed later) (Table 4).

Parameters of ICG disposition that were signi� -cantly different between Group 1 and Group 2 im-mediately before surgery included AUC, AUMC,CL, K10, K10HL, and MRT(Table 5). When Group 1baseline values were compared to values obtainedin that group immediately before surgery, no dif-ferences were noted. When Group 2 baseline val-ues were compared to values obtained in that groupimmediately prior to surgery, all parameters werefound to be signi� cantly different. The magnitude ofthese changes over time were: AUC, mean 73% de-crease (p < .0004); AUMC, mean 94% increase (p <

.003); CL, mean 68% decrease (p < .0001); CMAX,

TABLE 3 Temporal onset (weeks) of clinical laboratoryabnormalities in Group 2 dogs

Clinical laboratory Mean § SD MedianN abnormality (weeks) (weeks)

10 Increased PRBA 4.4 § 1.9 410 Increased POBA 4.4 § 1.9 410 Decreased PLAT 7.1 § 2.0 710 Increased POBSP 7.1 § 2.0 810 Prolonged PTT 7.5 § 2.6 710 Decreased ALB 7.5 § 3.1 810 Increased ALT 8.4 § 7.8 6.510 Increased ALKP 9.4 § 7.4 6.510 Increased PRNH3 10.2 § 5.3 1010 Decreased AT3 14.7 § 15.5 89 Decreased BUN 9.9 § 7.7 88 Decreased CHOL 8.6 § 3.3 87 Decreased PCV 7.9 § 0.9 85 Decreased TP 8.4 § 4.3 85 Prolonged PT 8.6 § 3.7 85 Increased GGT 13.4 § 4.4 113 Increased PONH3 20.3 § 17.0 113 Increased BILI 10.3 § 3.5 10

Note. PRBA, preprandial bile acids; POBA, postprandial bileacids; PLAT,platelets; POBSP, post sulfobromophthalein;PTT, partial thromboplas-tin time; ALB, albumin; ALT, serum alanine aminotransferase; ALKP,alkaline phosphatase; PRNH3, preprandial ammonia; AT3, antithrom-bin III; BUN, blood urea nitrogen; CHOL, cholesterol; PCV, packedcell volume; TP, total protein, PT, prothrombin time; GGT, gamma-glutamyltransferase; PONH3, postprandial ammonia; BILI, total biliru-bin.

mean 22% decrease (p < .0007); K10, mean 74%decrease (p < .0001); K10HL, mean 79% decrease(p < .0001); MRT, mean 79% decrease (p < .0001);and V d, mean 35% decrease (p < .008).

Abdominal Ultrasonography

Abnormal vessels near the left kidney, hyperechoicareas in the liver, and a decrease in number andsize of hepatic and portal veins within the hepaticparenchyma were visible ultrasonographically in dis-eased dogs (Figure 1). These changes occurred overtime and were most pronounced in diseased dogsimmediately prior to surgery. The temporal identi� -cation of PSS by ultrasonography is listed in Table 6.

Hepatic Scintigraphy

A progressive decrease in hepatic venous blood� ow was visible on hepatic scintigraphy in diseaseddogs. Control dogs maintained normal hepatic per-fusion indices. Diseased dogs had the highest hepatic

A MODEL OF MULTIPLE PORTOSYSTEMIC SHUNTING 49

New Methodologies

TABLE 4 Clinical laboratory test means and standard deviations in Group 2 that were signi�cantly different immediately beforesurgery as compared with baseline and as compared to Group 1 baseline

Group 1 ¡ baseline Group 2 ¡ baseline Group 2 ¡ before( N = 6), (N = 10), surgery (N = 10),

Variable mean § SD mean § SD mean § SD

PCV (%) 51.1 § 6.4 51.9 § 2.5 39.4 § 8.4a

ALT (U/L) 78.2 § 103.5 43.6 § 34.7 260.5 § 232.9a

ALB (g/dL) 3.1 § 0.2 3.3 § 0.3 2.2 § 0.4a

ALKP (U/L) 42.5 § 9.2 67.0 § 34.8 372.8 § 218.0a

BUN (mg/dL) 14.7 § 2.3 13.8 § 4.9 5.1 § 2.7a

PRBA (l mol/L) 6.9 § 3.2 2.6 § 3.3 105.5 § 66.7a

POBA (l mol/L) 8.2 § 5.5 7.6 § 6.8 209.8 § 89.7a

PRNH3 (l g/dL) 8.2 § 7.0 19.6 § 12.4 95.9 § 64.8a

PONH3 (l g/dL) 11.9 § 11.1 15.0 § 9.9 161.1 § 88.7a

PLAT ( £ 103/l L) 254.2 § 20.8 237.3 § 63.8 133.7 § 76.1a

PTT (s) 13.9 § 1.4 13.3 § 0.8 19.9 § 3.6a

AT3 (%NCP) 102.7 § 2.4 107.5 § 10.4 56.5 § 11.4a

POBSP (%) 2.2 § 3.7 5.6 § 5.5 37.0 § 12.6a

Note. N, number of observations; SD, standard deviation; PCV, packed cell volume; ALT, serum alanine aminotransferase; ALB, albumin; ALKP,alkaline phosphatase; BILI, total bilirubin; BUN, blood urea nitrogen; GLUC, glucose; PHOS, phosphorus; TP, total protein; GGT, gamma-glutamyltransferase; PRBA, preprandial bile acids; POBA, postprandial bile acids; PRNH3, preprandial ammonia; PONH3, postprandial ammo-nia; PLAT, platelets; PT, prothrombin time; PTT, partial thromboplastin time; AT3, antithrombin III; PRBSP, pre sulfobromophthalein; POBSP, postsulfobromophthalein.

aSigni�cant difference between Group 2 laboratory values seen immediately before surgery as compared to Group 1 and Group 2 baselinevalues.

perfusion indices (7.9 § 7.9) late in the course ofhepatic disease compared to baseline examinations(2.5 § 0.6) ( p § .05). Photographs of hepatic scinti-graphic examinations from representative animals ineach group are shown (Figures 2 and 3).

Surgery and Portography Findings

All dogs in Group 2 developed hepatic diseaseand multiple PSS as documented by exploratory la-

TABLE 5 Indocyanine green pharmacokinetic parameters seen in Group 2 dogs (baseline and immediately before surgery) ascompared to baseline of Group 1 dogs

Group 1 ¡ baseline Group 2 ¡ baseline Group 2 ¡ before( N = 6), (N = 10), surgery (N = 10),

Variable mean § SD mean § SD mean § SD

Vd (L/kg) 0.043 § 0.008 0.038 § 0.004 0.058 § 0.021a

K10 (min¡ 1) 0.080 § 0.017 0.064 § 0.018 0.017 § 0.013a, b

AUC (mg/ml/min) 150.24 § 12.12 235.26 § 125.26 886.17 § 455.58a, b

K10HL (min) 9.01 § 1.98 12.28 § 6.39 59.22 § 29.75a, b

CMAX (mg) 11.92 § 2.28 13.27 § 1.41 10.41 § 1.71a

MRT (min) 13.00 § 2.86 17.72 § 9.22 85.27 § 42.82a, b

AUMC (mg/ml/m2) 1970.85 § 576.73 5193.95 § 6807.90 92577.99 § 79127.89a, b

CL (L/min/m2) 0.0033 § 0.0003 0.0025 § 0.0007 0.0080 § 0.0006a, b

Note. SD, standard deviation; Vd, volume of distribution; K10, elimination rate constant; AUC, area under the curve; K10HL, elimination half-life;CMAX, maximum concentration; MRT, mean residence time; AUMC, area under the moment curve; CL, clearance.

aSigni�cant difference between Group 2 values seen immediately before surgery as compared to Group 2 baseline values.bSigni�cant difference seen in Group 2 values seen immediately before surgery as compared to Group 1 baseline values.

parotomy and portography (Figures 4 and 5). Shunt-ing vessels were most predominant in the regionof the left kidney, but anomolous connections be-tween the portal venous system and the caudal venacava and its tributaries were noted throughout theabdomen. Portoazygous shunts were also noted insome dogs on portography. Additionally, large di-lated vessels were noted in the falciform ligamentand greater omentum. Other surgical � ndings in-cluded enlarged mesenteric veins and an enlarged

50 L. M. HOWE ET AL.

New Methodologies

TABLE 6 Temporal identi�cation (weeks) of multipleportosystemic shunts by abdominal ultrasonographic exami-nation in each Group 2 dog

Dog number (group 2) Week shunts identi® ed

052 8059 4159 4250 4296 32300 9301 13303 4308 6335 15Mean § SD 9.9 § 8.7Median 7

portal vein in all Group 2 dogs. No abnormalitieswere noted in Group 1 dogs at exploratory laparo-tomy or portography.

Hepatic Histology

Histologically, hepatic biopsies from diseased dogsshowed moderate to severe, diffuse, chronic hepatic� brosis (n = 9), hepatic atrophy and regeneration

FIGURE 1 Abdominal ultrasound examination of a Group 2 dog demonstrating the presence of multiple PSS in the region of theleft kidney. Arrows denote the shunts.

(n = 8), and moderate to severe centrilobular necro-sis (n = 5) (Figure 6). No lesions were present inhepatic specimens from control dogs.

DISCUSSION

There are many different models to study cirrho-sis, in general, or to study speci� c types of human cir-rhosis [1, 2, 23–38]. The carbon tetrachloride modelhas been developed in both the dog and rat [23–27]. An advantage of this model is that it consis-tently produces cirrhosis. However, three disadvan-tages of this model have been reported. Rozga andcoworkers showed that treatment with phenobarbi-tal throughout the carbon tetrachloride inductionperiod is necessary for consistent production of hep-atic cirrhosis in rats [23]. Yet phenobarbital itselfalso induces hepatotoxicity in rats not consistentwith human hepatic cirrhosis [23], rendering the ap-plicability of the model questionable. More impor-tantly, lesions produced by carbon tetrachloride arenot speci� c to the liver, with changes occurring inthe spleen, pancreas, lymph nodes, monocytes, andkidney [23]. Lastly, several investigators have notedthat the lesions produced by carbon tetrachloride

A MODEL OF MULTIPLE PORTOSYSTEMIC SHUNTING 51

New Methodologies

FIGURE 2 Hepatic scintigraphy examination of a Group 2 dog illustrating uptake of the radioactive colloid material by organsother than the liver (arrows), which is typical in dogs with multiple PSS.

often don’t persist after discontinuation of the toxin[23, 24].

A second model used to induce hepatic cirrho-sis in rats and dogs is ligation of the common bileduct [28–32]. Advantages of this technique are thatit is technically easy to perform and does not require

FIGURE 3 Hepatic scintigraphy examination of a Group 1 dog illustrating normal uptake of the radioactive colloid material bythe liver, with minimal uptake by other organs.

constant monitoring of the animals during the de-velopmental stage of disease, as do techniques thatrequire toxin administration. A disadvantage of thistechnique is that it closely mimics only biliary cir-rhosis. Although this makes it a superior model forstudying secondary biliary cirrhosis, this disease is

52 L. M. HOWE ET AL.

New Methodologies

FIGURE 4 Dimethylnitrosamine-induced multiple PSS in a Group 2 dog as seen at surgery demonstrating numerous veins(arrows) connecting the portal venous system and the caudal vena cava.

uncommon in the dog, and the model may not beas good a model for studying types of cirrhosis thatare not caused by chronic biliary stasis.

Ferric nitrilotriacetate has been used to produce acirrhotic syndrome in the rat that is similar to pig-

FIGURE 5 An intraoperative portogram showing numerous anomalous connections (arrows) between the portal vein and thecaudal vena cava in a Group 2 dog.

ment cirrhosis (hemochromatosis) in humans [33].Nitrilotriacetate is a synthetic aminopolycarboxylicacid that ef� ciently forms water-soluble chelate com-plexes with iron and other metal cations at neutralpH. An advantage of this technique is that it appears

A MODEL OF MULTIPLE PORTOSYSTEMIC SHUNTING 53

New Methodologies

FIGURE 6 An hepatic biopsy specimen collected from aGroup 2 dog illustrating cellular degeneration, �brosis, andin�ammatory cellular in�ltrate.

to closely mimic hemochromatosis. The techniqueis not useful for studying other types of hepatic cir-rhosis, however.

The use of cupric nitrilotriacetate has been re-ported to produce a model of Wilson’s disease inthe rat [34]. The cupric nitrilotriacetate is admin-istered intraperitoneally in this model. This modelcould conceivably be used to study copper cirrho-sis in dogs as well. A disadvantage is that this tech-nique does not mimic other types of hepaticcirrhosis.

Thioacetamide is another substance that has beenused to produce cirrhosislike lesions in the rat [35–37]. In 1988, Muller et al. [35] reported on the modeland its reliability. The model produces a micronodu-lar type of cirrhosis. Using a standard dosage, thereis a 90% survival rate after 3 months of administra-tion. An advantage of this technique is that a stan-

dard dose tends to produce approximately similarlesions and severity of disease in most animals. Adisadvantage involves the length of time required toproduce disease, although it is similar to many of theother toxins used to produce cirrhosis. Another se-rious disadvantage of this model is that cirrhoticlikelesions only persist for about 2 months after toxindiscontinuation, requiring studies to be done duringthe � rst 2 months following toxin administration.

To avoid the disadvantages of inducing hepaticdisease using the methods just noted, dimethylni-trosamine was selected to induce hepatic disease inthis study. Dimethylnitrosamine has been used as amodel of canine and human cirrhotic hepatic disease[1, 2, 9]. Dimethylnitrosamine is an N -alkyl-N -nitro-so compound characterized by two major toxicities:hepatic necrosis with � brosis, and carcinogenicity[1, 2]. Intermittent administration of DMNA pro-duces progressive hepatic cirrhosis [1, 2]. The sever-ity of cirrhosis is dose and time dependent [1, 2].Advantages of the use of DMNA over other meth-ods include hepatic speci� city, similar mechanism ofaction to that seen in spontaneous diseases, and lackof resolution of cirrhotic hepatic disease. Disadvan-tages of this method include toxicity of DMNA tohumans, variability in response to DMNA by the in-dividual dog, and need for repeated administrationto produce hepatic disease. Despite these disadvan-tages, DMNA would appear to produce hepatic dis-ease that is more similar to the spontaneous diseasestate than other methods of inducing hepatic disease.

Under physiologic conditions, DMNA is a che-mically stable compound that requires hepaticenzymatic transformation to active intermediates fortoxicities to be manifest [3, 4]. The mechanisms ofDMNA toxicity in carcinogenesis and hepatic necro-sis with � brosis appear to involve alkylation of intra-cellular nucleophilic macromolecules (e.g., protein,RNA, or DNA, especially those bound by the endo-plasmic reticulum) by a reactive metabolite (carbo-nium ion) that is released after oxidative metabolismof DMNA by cytochrome P-450 [3, 4, 39]. Thismetabolic transformation appears to be extensive.Amino acid incorporation into proteins is decreasedwithin hours after DMNA administration [12]. Thisresults in signi� cant depression of hepatic proteinsynthesis [12].

54 L. M. HOWE ET AL.

New Methodologies

Dimethylnitrosamine has been used to induce ei-ther hepatic necrosis or carcinogenicity. The com-pound is capable of producing severe hepatic necro-sis in many species [5]. Regardless of the route ofadministration, DMNA toxicity is speci� c for theliver. In 1970, Madden and co-workers described theproduction of cirrhotic hepatic disease in the dog us-ing DMNA [2]. In that study lesions were producedusing a low dose (2.5 l l/kg or 2.5 mg/kg) several daysa week for 4 to 6 weeks. Several routes of administra-tion of the drug could be used: orally with or withoutfood, or by direct infusion into the portal vein. Al-though the severity of the disease and the histologiclesions were not consistent in all dogs studied, thelesions were often typical of cirrhosis and were ac-companied by portal hypertension and PSS in someanimals. In Madden’s study some animals had mildhistologic lesions that tended to resolve with time,and some animals progressed to cirrhotic hepatic dis-ease. Subsequent authors showed that when dogswere given DMNA orally with food (2 mg/kg) for2 consecutive days a week for 1 month, results werevariable among dogs [9, 38]. Approximately 30% ofdogs died due to acute fulminant hepatic disease,20–30% died from chronic hepatic disease, and 30%of dogs developed cirrhotic hepatic disease about 31weeks after the start of DMNA administration. Tenpercent of dogs did not develop any form of hepaticdisease. Once produced, cirrhotic lesions generallydid not resolve (as compared to other types of toxin-induced cirrhotic disease). Boothe et al. showed thatthree stages of progressive hepatic disease (mild, mod-erate, and severe) could be produced by careful mon-itoring and titration of DMNA dosage based on theindividual dog’s response [1]. Attrition rate of dogscould also be decreased using this approach. How-ever, control over which stage of hepatic disease anindividual dog developed was lacking. In the presentstudy, multiple acquired PSS could consistently beproduced in dogs using careful monitoring and in-dividualization of DMNA treatment regimens.

Clinical signs seen in Group 2 dogs of this studywere mild prior to surgery and were consistent withhepatic disease. Intermittent anorexia and fever wereoften the � rst clinical signs noted, with ascites notednext. Ascites was easily manageable by a sodium-restricted diet. Weight loss was most pronounced im-

mediately before surgery. Two dogs developed icterusthat was indicative of pronounced cholestatic dis-ease. Toxin administration was discontinued imme-diately once icterus was noted.

Hepatic disease was characterized by increases inPRBA and POBA (13- to 54-fold), PRNH3 (2- to 5-fold), and BSP retention times (20- to 40-fold) notedimmediately presurgically. Plasma ICG clearance de-creased 68% compared to baseline. Changes were� rst observed in PRBA and POBA. Additional use-ful tests in monitoring the development of hepaticdisease included ALB (mean 33% decrease), BUN(mean 63% decrease), ALT (mean 83% increase),ALKP (mean 82% increase), PCV (mean 25% de-crease), PLAT (mean 44% decrease), PTT (mean 35%increase), and AT3 (mean 48% decrease).

Clinical signs, clinical pathology testing, abdomi-nal ultrasound, and hepatic scintigraphy � ndings indogs of this study are similar to spontaneously oc-curring hepatic disease with multiple PSS [40]. Dogswith experimentally induced hepatic disease and PSSand those with spontaneously occurring hepatic dis-ease and PSS both had similar clinical signs, includ-ing reduced weight, decreased appetite, and ascites.Fever and icterus, however, were not recorded in dogswith spontaneous disease. Clinical pathology � nd-ings were also similar among dogs with experimen-tally induced and spontaneously occurring disease.Further, abdominal ultrasound and hepatic scinti-graphy � ndings were similar among dogs with spon-taneous and DMNA-induced hepatic disease. Thus,it would appear that the experimental model usedin this study is an excellent model of spontaneouslyoccurring hepatic disease and multiple PSS.

CONCLUSIONS

Clinical signs observed before surgery in diseasedanimals of this study were mild, responsive to symp-tomatic therapy, and consistent with those seen inother studies using DMNA as an hepatotoxin. Se-vere clinical hepatic disease was avoided in diseaseddogs in this study prior to surgery by carefully moni-toring and titrating the DMNA dose. Shunt locationand clinical pathologic � ndings in diseased animalswas similar to that seen in spontaneously occurringmultiple PSS [40].

A MODEL OF MULTIPLE PORTOSYSTEMIC SHUNTING 55

New Methodologies

Based on this study, DMNA-induced PSS seemsto be an appropriate model for spontaneously oc-curring multiple PSS secondary to portal hyperten-sion in the dog. Dimethylnitrosamine-induced PSSmay also prove to be a useful model for studyinghepatic cirrhosis, portal hypertension, and portosys-temic shunting in humans.

REFERENCES

1. Boothe DM, Jenkins WL, Green RA, Corrier DE, CullenJM, Boothe HW, Weise D. Dimethylnitrosamine-inducedhepatotoxicosis in dogs as a model of progressive caninehepatic disease. Am J Vet Res. 1992;53:411–420.

2. Madden JW, Gertman PM, Peacock EE. Dimethyl-nitrosamine-induced hepatic cirrhosis: A new caninemodel of an ancient human disease. Surgery. 1970;68:260–268.

3. Lake BG, Collins MA, Harris RA, Phillips JC, Cottrell RC,Gangolli SD. Studies on the metabolism of dimethylni-trosamine in vitro by rat liver preparations. I. Compari-son with mixed function oxidase enzymes. Xenobiotica.1982a;12:435–445.

4. Kroeger-Koepke MB, Koepke SR, McClusky GA, MageePN, Michejda CJ. Alpha-hydroxylation pathway in the invitro metabolism of carcinogenic nitrosamines: N-Nitro-sodimethylamine and N-nitroso-N-methylaniline. ProcNatl Acad Sci USA. 1981;78:6489–6493.

5. Magee PN, Barnes JM. Carcinogenic nitroso compounds.Adv Cancer Res. 1967;10:163–247.

6. Mirvish SS. N-Nitroso compounds: Their chemical and invivo formation and possible importance as environmen-tal carcinogens. J Toxicol Environ Health. 1977;2:1267–1277.

7. Matsuda Y, Matsumoto K, Yamada A, Ichida T, Asakura H,Komoriya Y, Nishiyama E, Nakamura T. Preventative andtherapeutic effects in rats of heaptocyte growth factorinfusion on liver �brosis/cirrhosis. Hepatology .1997;26:81–89.

8. George J, Chandrakasan G. Glyocprotein metabolism indimethylnitrosamine induced hepatic �brosis in rats. Int JBiochem Cell Biol. 1996;28:353–361.

9. Testas P, Benichou J, Perrin N, Vieillefond A, BenhanouM. Comparative study of two models for experimentalcirrhosis in the dog. Eur Surg Res. 1978;10:146–152.

10. Ryhanen L, Stenback F, Ala-Kokko L, Savolainen ER.The effect of malotilate on type III and type IV colla-gen, laminim and �bronectin metabolism in dimethyl-nitrosamine-induced liver �brosis in the rat. J Hepatol.1996;24:238–245.

11. Preussman R, Weissle M. The enigma of organ-speci�city of carcinogenic nitrosamines. Trends Pharm Sci.1987;8:185–189.

12. Mukherjee T, Gustafsson RG, Afzelius BA, Arrhenius E.Effects of carcinogenic amines on amino acid incorpora-tion by liver systems. II. A morphologic and biochemical

study on the effect of dimethylnitrosamine. Cancer Res.1963;23:944–953.

13. Korsrud GO, Grice HG, Goodman TK, Knipfel JE,McLaughlan JM. Sensitivity of several serum enzymesfor the detection of thioacetamine-, dimethylnitrosamine-and diethylolamine-induced liver damage in rats. ToxicolAppl Pharmacol. 1973;26:299–313.

14. Butler-Howe LM, Boothe HW, Boothe DM, Laine GA,Calvin JA. Effects of vena caval banding in experimen-tally induced multiple portosystemic shunts in dogs. AmJ Vet Res. 1993;54:1774–1783.

15. Howe LM, Boothe DM, Boothe HW. Endotoxemia associ-ated with experimentally induced multiple portosystemicshunts in dogs. Am J Vet Res. 1997;58:83–88.

16. Boothe DM, Brown SA, Jenkins WL, Green RA, CullenJM, Corrier DE. Indocyanine green disposition in healthydogs and dogs with mild, moderate, or severe dimethyl-nitrosamine-induced hepatic disease. Am J Vet Res.1992;53:382–388.

17. Wrigley RK, Konde LJ, Park RD, Lebel JL. Ultrasonographicdiagnosis of portocaval shunts in young dogs. J Am VetMed Assoc. 1987;191:421–424.

18. Koblik PD, Hornoff WJ, Breznock EM. Quantitative hepaticscintigraphy in the dog. Vet Radiol. 1983;24:226–231.

19. Koblik PD, Hornoff WJ, Breznock EM. Use of quantita-tive hepatic scintigraphy to evaluate spontaneous por-tosystemic shunts in 12 dogs. Vet Radiol. 1983;24:232–236.

20. Moon ML. Diagnostic imaging of portosystemic shunts.Semin Vet Med Surg (Small Anim). 1990;5:120–126.

21. Butler LM, Fossum TW, Boothe HW. Surgical managementof extrahepatic portosystemic shunts in the dog and cat.Semin Vet Med Surg (Small Anim). 1990;5:127–133.

22. Whiting PG, Peterson SL. Portocaval shunts. In: Slatter D,ed. Textbook of Small Animal Surgery (2nd ed). Philadel-phia: WB Saunders; 1993:660–677.

23. Rozga J, Foss A, Alumets J, Ahrens B, Jeppsson B,Bengmark S. Liver cirrhosis in rats: Regeneration andassessment of the role of phenobarbital. J Surg Res.1991;51:329–335.

24. Doi K, Kurabe S, Shimazu N, Inagaki M. Systemichistopathology of rats with CCl4-induced hepatic cirrho-sis. Lab Anim. 1991;25:21–25.

25. Torres EM, Bouza JIP, Hernandez MMA, Martin MJA, BravoAL. Experimental carbon tetrachloride-induced cirrhosisof the liver. Int J Tiss Reac. 1988;10:245–251.

26. Mizumoto M, Arii S, Furutani M, Nakamura T, IshigamiS, Monden K, Ishiguro S, Fujita S, Imamura M. NO as anindicator of portal hemodynamics and the role of iNOS inincreased NO production in CCl4-induced liver cirrhosis.J Surg Res. 1997;70:124–133.

27. Mion F, Geloen A, Agosto E, Minaire Y. Carbontetrachloride-induced cirrhosis in rats: In�uence of theacute effects of the toxin on glucose metabolism. Hep-atology. 1996;23:582–588.

28. Koblik PD, Hornoff WJ, Yen C, Komtebedde J, BreznockE, Fisher P. Comparison of shunt fraction estima-tion using transcolonic iodine-123-iodoamphetamine andtechnetium-99m-pertechnetate in a group of dogs with

56 L. M. HOWE ET AL.

New Methodologies

experimentally-induced chronic biliary cirrhosis. J NucMed. 1991;32:124–129.

29. Morgan TR, Morgan K, Jonas G, Thillainadarajah I. Atrialnatriuretic factor in experimental cirrhosis in rats. Gas-troenterology. 1992;102:1356–1362.

30. Oberti F, Pilette C, Rif�et H, Maiga MY, Moreau A,Gallois Y, Girault A, le Bouil A, Le Jeune JJ, Saumet JL,Feldmann G, Cales P. Effects of simvastatin, pentoxi-fylline and spironolactone on hepatic �brosis and por-tal hypertension in rats with bile duct ligation. J Hepatol.1997;26:1363–1371.

31. Criado M, Flores O, Ortiz MC, Hidalgo F, Rodriguez-LopezAM, Eleno N, Atucha NM, Sanchez-Rodriguez A, ArevaloM, Garcia-Estan J, Lopez-Novoa JM. Elevated glomeru-lar and blood mononuclear lymphocyte nitric oxide pro-duction in rats with chronic bile duct ligation: Role ofinducible nitric oxide synthase activation. Hepatology .1997;26:268–276.

32. Wong J, Zhang Y, Lee SS. Effects of portacaval shuntinghyperdynamic circulation in bile duct-ligated cirrhotic rats.J Hepatol. 1997;26:369–375.

33. Awai M, Narasaki M, Yamanoi Y, Seno S. Induction ofdiabetes in animals by parenteral administration of fer-ric nitrilotriacetate: A model of experimental hemochro-matosis. Am J Pathol. 1979;95:663–674.

34. Toyokuni S, Okado S, Hamazaki S, Fujioka M, Li JL,Midorikawa O. Cirrhosis of the liver induced by cupricnitrilotriacetate in Wistar rats: An experimental model ofcopper toxicosis. Am J Pathol. 1989;134:1263–1274.

35. Muller A, Machnik F, Zimmerman T, Schubert H.Thioacetamide-induced cirrhosis-like liver lesions in rats—Usefulness and reliability of this animal model. Exp Pathol.1988;34:229–236.

36. Buko V, Lukivskaya O, Nikitin V, Kuryan A, Dargel R. An-tioxidative effect of prostaglandin E2 in thioacetamide-induced liver cirrhosis. Exp Toxicol Pathol. 1997;49:141–146.

37. Torres MI, Fernandez MI, Gil A, Rios A. Effect of dietarynucleotides on degree of �brosis and steatosis induced byoral intake of thioacetamide. Dig Dis Sci. 1997;42:1322–1328.

38. Terpstra OT, van Vroonhoven TJ, van Vroonhoven MV,Noordhoek J, ten Kate FJ, Wilson JH. Temporary bene-�cial effect of arterialization of the liver in cirrhotic dogswith portocaval shunt. Eur Surg Res. 1982;14:333–343.

39. Grilli S, Prodi G. Identi�cation of dimethylnitrosaminemetabolites in vitro. Jpn J Cancer Res. 1975;66:473–480.

40. Boothe HW, Howe LM, Edwards JF, Slater MR. Multiple ex-trahepatic portosystemic shunts in dogs: 30 Cases (1981–1993). J Am Vet Med Assoc. 1996;208:1849–1854.

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