~ ~ ~ "';I"d- 3'.? .?c 'h 'f l~~i~~ C 1986 S. Karger AG, Base! 0014-312X/86/0~5-0283$2. 75/0 Eur. surg.Res.18: 283-292:(1986) A Predictable Pathophysiological Model of Porcine Hepatic Failure R. Hickman, M.H. Alp MRC Liver Research Group, Departments ofMedicine and Surgery, University ofCape Town and the Gastrointestinal Clinic, Groote Schuur Hospital, Observatory, South Africa Key Words. Pig .Experimentalliver failure .Carbon tetrachloride .Ischaemia Phenobarbitone .Amino acids Abstract. A technique is described in which acute hepatic failure may be uniformly induced in pigs using a combination of oral phenobarbitone pretreatment and 2-hour inter- ruption of hepatic arterial blood supply followe;d by intragastric- carbono tetrachloride. This results in deep hepatic coma ayer a narrowly predictable time span and death within 12-52 h. Amino acid leyels in plasma and cerebrospinal fluid oqtained 24 h after the induction of hepatic failure were similar tothose reported in human and experimental encephalopathy. The anhepatic models of Hickman et al. [11] and Herlin et al. [10] and the combined porta-caval shunt/hepatic artery ligation model arDe Groot et al. [6] sutTered from the defect of excluding the damaged liver from the circulation. Hickman et al. have found the thermochemotherapeutic rat model of Miyazaki et al. [17] to be unreliable and not reproducible in the pig (unpublished obser- vations) as has been the administration of galactosamine. Acetaminophen which pro- duces hepatic necrosis in hamsters [3] was proved unreliable in pigs [15]. Yellow phos- phorus has a predictably lethal etTect in dogs lntroduction Acute hepatic failure remains a potentially lethal complication of viTal or drug-induced hepatitis and methods of treatment are still unsatisfactory in general. While the use of galactosamineby Blitzer et al. [1] in the rab- bit and by Decker and Keppler [5] in the rat has resulted in adequatesmall animal models of experimental liver failure, there is still a need for a suitable large animal version to allow for repeated sampling or for the appli- cation of methods of treatment such as resin perfusion, plasma exchange, etc.
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C 1986 S. Karger AG, Base!0014-312X/86/0~5-0283$2. 75/0Eur. surg. Res. 18: 283-292:(1986)
A Predictable Pathophysiological Model of Porcine HepaticFailure
R. Hickman, M.H. AlpMRC Liver Research Group, Departments ofMedicine and Surgery, University ofCape Town and theGastrointestinal Clinic, Groote Schuur Hospital, Observatory, South Africa
Abstract. A technique is described in which acute hepatic failure may be uniformlyinduced in pigs using a combination of oral phenobarbitone pretreatment and 2-hour inter-ruption of hepatic arterial blood supply followe;d by intragastric- carbono tetrachloride. Thisresults in deep hepatic coma ayer a narrowly predictable time span and death within 12-52 h.Amino acid leyels in plasma and cerebrospinal fluid oqtained 24 h after the induction ofhepatic failure were similar tothose reported in human and experimental encephalopathy.
The anhepatic models of Hickman et al.[11] and Herlin et al. [10] and the combinedporta-caval shunt/hepatic artery ligationmodel arDe Groot et al. [6] sutTered from thedefect of excluding the damaged liver fromthe circulation. Hickman et al. have foundthe thermochemotherapeutic rat model ofMiyazaki et al. [17] to be unreliable and notreproducible in the pig (unpublished obser-vations) as has been the administration ofgalactosamine. Acetaminophen which pro-duces hepatic necrosis in hamsters [3] waspro ved unreliable in pigs [15]. Yellow phos-phorus has a predictably lethal etTect in dogs
lntroduction
Acute hepatic failure remains a potentiallylethal complication of viTal or drug-inducedhepatitis and methods of treatment are stillunsatisfactory in general. While the use ofgalactosamine by Blitzer et al. [1] in the rab-bit and by Decker and Keppler [5] in the rathas resulted in adequate small animal modelsof experimental liver failure, there is still aneed for a suitable large animal version toallow for repeated sampling or for the appli-cation of methods of treatment such as resinperfusion, plasma exchange, etc.
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284 Hickman/ Alp
butremains dangerous to its users even afteradministration to the animal [2]. Thus thesemethods did not fulfil completely the criteriasuggested by Terblanche et al. [23] whichstress not only the need for a large animalwith death from liver failure using methodswith minimal hazard to personnel but al soreproducibility and potential reversibility.To these five criteria could be added a sixth,videlicet reasonably long time interval fromonset of acute failure to death which wouldallow for the initiation of methods of treat-mento Thus in one way or another t~ese var-ious methods were unsatisfactory. A suitablemodel in the pig which fulfils these criteriahas been achieved by a combination of pre-treatment with phenobarbitone followed bytimed reversible hepatic artery occlusion anda graded oral dose of carbon tetrachloride asdescribed in 1974 by van Leenhoffet al. [25].Hepatic failure and coma occurred predict-ably within narrow time limit~ and were in-variably followed by death. Plasma and cere-brospinal fluid (CSF) free amino acids wereassayed before and after the induction ofhepatic coma.
I Methods
Large White pigs, 6-8 weeks old weighing 15-20 kg were used throughout. Four groups of animalswere used which had been starved for 24 h (table 1).Group I (10 pigs) was given 0.5 ml/kg oral carbon tet-rachloride (CCI4) alone. Group 11 (16 pigs) was given0.5 ml/kg CCI4 followed by 2 h of hepatic artery liga-tion 1-3 days later. Group 111.( 1 O pigs) was treated for3 days with 8 mg/kg phenobarbitone prior ~o hepaticartery ligation for 2 h followed immediately by0.5 ml/kg CCI4. Group IV (8 pigs) was treated in asimilar fashion as group 111 but prior administration ofphenobarbitone was omitted. In all groups CCI4 wasinstilled into the stomac;-h by gastric tube or direct
injection.
For groups I and 11 CCI4 was given after a smalIdose of intravenous sodium pentothal to induceanaesthesia. Anaesthesia for hepatic artery Iigation ingroups 11, 111 and IV was induced with 1-3 mg/kgsodium pentothal and maintained with oxygen andnitrous oxide. AlI pigs were intubated for any proce-dure to prevent puImonary contamination with
CCI4.At laparotomy, the hepatic artery was dissected
free in the porta hepatis. After all ligamentous andvascular connections passing the diaphragm had beendivided, the hepatic artery above the right gastricbranch afilie coeIiac artery was occluded with a snareofpolyethylene tubing. The abdomen was closed witha single layer of DermaIon. Two hour~ Iater the ani-mals were reopened rapidly under minimal nitrousoxide anaesthesia, the snare was removed and the pul-sation ofthe hepatic artery was confirmed. Then CCI4was injected directIy into the stomach. The abdomenwas closed in layers. Cathéters (8 Fr.) were placed intothe femoraI artery and vein for measurement ofbIoodpressure, blood sampling and infusion. Intravenousfluids were given to maintain urine output, and thepigs were housed in individual warmed cages. Bloodand CSF samples were taken pre-operatively, imme-diately after the ligation and after 24 and 48 h if theanimal survived. Postmortem examination was car-ried out in all pigs and sampIes ofliver and lung .wereexamined histologicaIly.
Serum bilirubin and serum glutamate oxaloacetatetransaminase (SGOT) were estimated by standardtechniques. Prothrombin index was estimated by amanual Quick one-stage method using a Dade stan-dard kit containing rabbit brain thrombopIastin.Plasma ammonia le veIs were estimated using a Hy-land blood ammonia test kit. The samples were col-lected directly into heparin-containing tubes on ice,centrifuged within 30 min, stored at 20.C and testedwithin 48 h of collection. Samples were measured induplicate and compared with known standards sup-plied with the kit.
CSF was obtained by lumbar puncture from pigs ingroup 11, 111 and IV. Sgme animals needed 2-3 mIsodium pentothaI as a light anaesthetic for this proce-dure but many were so deeply unconscious that thiswas not necessary. This waj taken as additional evi-dence of encephaIopathy. Amino acid concentrationsin the serum and CSF both before and after inductionofhepatic faiIure were estimated using a Waters HPLCreverse-phase C-I8 coIumn (3.9 mI X 30 cm) with a
285Model of Hepatic Failure
Table l. Survival fol1owing induction ofliver failure
SurvivalGroup Method of induction
CC!4 0.5 rn!/kghepatic arteria! ischaernia for 2 h
!-3 days !ater
11(n = 16)
short-term surviva1 (15-52 h)phenobarbitone, 8 rng/kg for 3 dayshepatic arterial occlusion for 2 hCCl4 0.5 rnl/kg
postoperative deathpneumoniashort-term survival>10 days 3
l~
linear acetonitrile gradient (10-55%) over 32 min in15 mM phosph~te buffer (pH 7.2) [12]. The equip-ment consisted of two Waters Model 6000 A pumps,a Waters -Model 680 automatic gradient controllerand a Wisp 710 B injector. Precolumn derivation andfluorescente were obtained by using ethanethiol ando-phthaldialdehyde [14], the eluted amino acids beingidentified and quantitated using a Cecil continuous-flow UV monitor set at a wavelength of 330 nm and0.02 absorbance by their retention times and absor-bance peaks on the column as read by a WatersModel M730 Data Module. Homocysteic acid(10 mM) was used as an internal standard. Concen-trations of internal standard were as follows: 5 ~ fornorIrlal CSF, 7.5 ~ for norIrlal plasma and 10~ forhepatic failure plasma and CSF. Two hundred mi-croliters of plasma or CSF was always derived but theamount of derived plasma applied to the column alsovaried: 15 ~ for hepatic failure; 25 ~ for norIrlalplasma and hepatic failure CSF and 50 ~ for norIrlalCSF. The method is limited by the similar retentiontimes of glycine and threonine which are therefore
Results
Survival (table 1)Group lo Of the 10 pigs given 0.5 ml/kg
CCI4 via a gastric tube under light pentothalanaesthesia, 2 died 8 h later without regain-ing consciousness and another died from se-'veTe pneumonic consolidation after 36 h.The remainder lived for 10 days before sacri-fice with no evidence of coma. AII showedhistological features of mild hepatic damageat post-~ortem (fig. la).
Group 11. Of the 16 pigs givén 0.5 ml/kgCCI4 1-3 days before hepatic artery occlu-sion, 4 died before operation and 2 died dur-ing the operation from excessive bleeding. Ofthe remaining lO, 3 were still alive after 1week and were sacrificed, while 7 survivedfrom 12 h to 6 days (average 3.1 days). The
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287Model of Hepatic Failure
Table 11. Biochemical changes in porcine induced. liver failure
Group 111(n = 10)
Group IV(n = 8)
Group II(n = 16)
Normal(n = 45)
Test
Arnrnonia 75(53-100)
Prothrombin index
Figures in parentheses indicate range.* p < 0.05; ** p < 0.0 1; *** P < 0.00 I
postmortem. Pulmonary consolidation wasnot seen although on occasion the lungs weredotted with 2-4-mm haemorrhagic aTeaS.The .livers in all pigs were pale and swollen.Histology showed severe centrilobular necro-sis typical of CCl4 poisoning (fig. lb). Noother intraabdominallesion was found.
Group IV. Eight pigs underwent 2 h ofhepatic artery ligation followed by intragas-tric CCl4 (0.5 ml/kg) without prior phenobar-bitone treatment. Three died within 36 h, 2from respiratory failure and 1 from wounddehiscence. One died from an overdosage ofsodium pentothal used to induce anaesthesiafor lumbar puncture at 24 h, 1 from no ob-vious cause at 48 h, and there were 3 long-term survivors. As for group 1, the hepatichistological changes were variable but gener-ally mildo
reduced response to painful stimuli beforeregaining consciousness. Histologically allshowed evidence of varying degrees of he-patic necrosis at postmortem.
Group III~ Ten pigs were given 8 mg;kgphenobarbitone orally for 3 days followed by2 h of hepatic artery ligation and intragastricCCl4 (0.5 ml/kg). All 10 animals recoveredfrom the procedure" and were alert but notstanding 6 h postoperatively. Death occurredin all pigs within 15-52 h (average 32 h). Thiswas preceded by 6-36 h of deep hepaticcoma, the pigs being unconscious and onlyslightly responsive to painful stimuli.
Death was frequently preceded by twitch-ing and convulsions. No evidence ofintrace-rebral or CSF infection could be found at
:
Fig. 1. a Biopsy from a pig sacrificed 4 days afteradministration of oral CCl4 alone. Note thé minimalpericentral destruction. HE. X 40. b A biopsy from apig from group 1II (oral phenobarbitone, hepatic ar-terial ischaemia and intragastric CCI4) showing severecentrilobular necrosis destroying two thirds of the
hepatic lobule. HE. X 40.
Biochemical Resulls (table II)There were no significant biochemical
changes in group l. Levels of SGOT in groupII pigs 24 h postoperatively were moderatelyelevated compared with those obtained pre-
Amino Acid Changes (tables III, IV)There were no significant differences in
plasma or CSF amino acid levels in group I(CCI4 treated) pigs as compared with preop-erative values obtained from 16 animals.
There were significant elevations of gIuta-mine, arginine and methionine in CSF ofgroup 11 pigs, while in group m animals, theamino acids were even more abnormal, withonly gIutamic acid, aspa!agine, serine andomithine failing to- show statistically signifi-cantrises. AlthougÍt "branched-chain and aro-matic amino acids as well as methionine weresignificantly elevated~ t'\te greatest rise was inthe latter two groups, markedly lowering thebranched-chain:aromatic amino acid ratio.This was common both to the plasma and theCSF.
operatively but fell rapidly to be within nor-mallimits within 48-72 h. Blood ammoniawas not significantly elevated at any time norwas serum bilirubin.
In group 111 there were highly significantchanges in all samples studied. In 2 pigs sur-viving for longer than 48 h there was a fur-ther rise in bilirubin levels at the time with a50 % fall in ammonia and SGOT levels. Pro-thrombin indices fell to a mean of 45 % (nor-mal >80%).
Group IV pigs showed a moderate yet sta-tistically significant rise in SGOT levels 24 hafter operation which persisted for 48-72 h.No significant rise occurred in ammonia orbilirubin levels although the latter rase fur-ther in the subsequent 48 h. Prothrombin in-dex was not recorded.
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Model of Hepatic Failure 289
Table IV. CSF amino acid levels ~ol/l) in induced hepatic damage (mean :t SEM)
In group IV pigs, apart from a rise in CSFglutamine, Done of the amino acids showed
any significant change.Changes in plasma amino acids in groups
11 and IV were not significant but in group111, all aromatic amino acids and methioninewere markedly elevated (p < 0.001) but thebranched-chain amino acid levels remainedwithin normallimits.
Discussion
Many ofthe previous large animal modelsof hepatic failure have not been predictableor were irreversible because the damagedliver had been excluded from the circulation,
e.g. portacaval shunt with irreversible arterialischaemia as described by De Groot et al. [6]or the partial hepatectomy technique of Mi-nato et al. [16]. The present model is theoret-ically reversible because the damaged liverremains in the circulation and both insultsare of a sublethal nature (the dose of CC14 asdescribed by van Leenh<;>ff et al. [25] and thearterial ischaemia chosen fiom the experi-ence of Kahn et al. [13]). In further prelimi-nary studies, the administration of PGF2aresuted in survival in about 40% ofañi-mals, thus confirming the possibility of re-
versibility.Amino acid profiles for plasma have
been published for rabbits [4], rats [7], dogs[20], pigs [18] and humans [19]. Our resultsshow little variations from the spectropho-
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amounts of glycine and branched-chainamino acids, but not sufficiently to alter lheirratio compared to the aromatic amino acids.It is recognised that CSF obtained by lumbarpuncture may not be representative of thatfrom cisternal puncture but the hazards ofinfection by the latter method are greater. InOUT experiments, too, pre- and postoperativesamples were obtained by the same route.The in crease in plasma tryptophan in pigs ofgroup 111 and in CSF arginine may representonly species difference or be specific featuresof this method of induction of encephalopa-thy. Further comment could be made if val-ues were available from other models of en-cephalopathy in pigs.
The lack of correlation between aminoacid chariges in plasma and CSF needs com-mento In pigs of groups 11 and 111, apparentlygreater changes occurred in the levels in CSF,suggesting a defect in blood brain transport,despite the assertions of Herlin et al. [9] tothe contrary.
The pig model of phenobarbitone treat-ment in conjunction with timed hepatic ar-tery ligation and oral CCl4 regime has beenshown to fulfil all the criteria expressed byTerblanche et al. [23] except the possibilityof reversibility. In preliminary studies withthis model, administration of prostaglandinF2 resulted in a survival rate of about 40%for > 10 days, while administration ofamino acids, cholestyramine, cysteamine orglutathione proved unsuccessful [Alp, sub-mitted for publicati?n].- Thus, the model asmentioned earlier does appear to be revers-ible. We are also ~ble 10 demonstrate thatthe model fulfils the:'Sixth criteria of a rea-sonable time interval between induction offailure and death, and thus it should proveuseful in further studies of therapeutic mo-dalities.
tometric assays in any species apart fromlower glutamine levels. Normal plasma leu-cine and valiDe levels in the pig are twicethose in human and rabbit plasma but otheressential amino acid concentrations are sim-ilar. However, little is known about theamino acid levels in pig plasma and CSFfollowing induction of hepatic failure. In a.limited study of plasma amino acids in pigssubjected to portacaval shunt and hepaticartery ligation, Wustrow et al. [26] founddepressiOI1 of the levels of branched-chainamino acids and elevation of aromaticamino acid levels. OUT results confirm therise in most plasma amino acids, particu-larly the aromatic amino acids, noted in hu-man hepatic failure [8] and hepatectomiseddogs [20]. However, branched-chain aminoacids were not significantly elevated in OUTstudy, resulting in a fall in the branched-chain:aromatic amino acid ratio, which issuggested to be important in the develop-ment of hepatic encephalopathy [8]. Thislack of change may relate specifically to ibisform of induction of hepatic failure, andpossibly contributory factors such as the in-sulin:glucagon ratio need to be investigatedbefore further interpretation.
The normal porcine CSF amino acids dif-fer from human levels [24] in that normalporcine CSF contains much greater amountsof asparagine, glycine and alanine, and re-duced arginine. However, the essentialamino acid concentrations are similar. In he-patic coma, the CSF profile changes mark-edly with the prominent abnormality beingin glutamine levels. CaTe was taken to ex-clude intracerebral infection in view of thereported changes in CSF amino acids in itspresence [22]. In comparison to amino acidlevels found in human hepatic encephalopa-thy [21], porcine CSF contained greater
Acknowledgements
Financial assistance was received from the Medi-cal Research Council of South Africa and the StaffResearch Fund of the University of Cape Town. Ms.Rose Innes and H. McLeod performed the biochemi-cal and amino acid analyses and Mrs. Grunwald pre-pared the histological specimens. Mrs. Johns typed themanuscript. Pre- and postoperative care was given bythe staff of the J .S. Marais Surgical Laborntory, espe-cially D. Tango.
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26 Wustrow, "f.; Hoom-Hickman, R. van; Hoom,W.A. van; Vinik, A.I.; Fischer, M.; Terblanche, J.:Acute hepaticischaemia in the pig -the changes inplasma hormones, amino acids and brain chemis-try. Hepato-Gastroenterology 28: 143 (1981).
Received: October 9, 1985.Accepted: November 27, 1985
Dr. R. Hickman,Department of Surgery,University of Cape Town,Observatory 7925 (South Africa)
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