Experimental models for hepatic encephalopathy

INTRODUCTION Al hough he e a e many models o animal es ing, he ideal model o ch onic li e disease in ol ing hepa ic encephalopa hy has no been desc ibed ye . Di e en p ob- lems associa ed wi h he models ha e led esea che s o de elop hei own, some imes unique expe imen al models, which makes di icul he compa ison be ween he esul s o he conduc ed s udies (1). Hepa ic encephalopa hy (HE) de ini ion, nomencla u e, diagnosis and quan i ica ion con- sensus we e published in 2002 (2) whe e h ee ypes o HE we e conside ed: ype A, associa ed wi h acu e li e ailu e, ype B, associa ed wi h he exis ence o po o-sys emic communica ion (by-pass) wi hou in insic li e disease and ype C, associa ed o li e ci hosis. HE ype C, in u n, is classi ied acco ding o hei o m o p esen a ion as spon- aneous o episodic HE, in ela ion o p ecipi a ing ac o s, pe sis en HE is subdi ided in o mild (HE g ade I), se e e (HE II-IV) o ea men -dependen (ea ly de eloped a e aboli ion o ea men ) and inally, he minimal HE, as he i s mani es a ion o HE. HE ype C is he mos common o m and om a clinical poin o iew, he episodic HE ype because o li e ci hosis decompensa ion is he mos ypical and ele an . The ideal model o HE should ep oduce mos o he clin- ical ea u es o his synd ome in humans, as occu ing mainly in pa ien s wi h ch onic li e disease, being p ecipi a ed by de ined ac o s, i should be e e sible wi h he co ec ion o p ecipi a ing ac o s and imp o ed using i aximin o ammo- nia lowe ing-d ugs, being s ongly associa ed wi h al e ed ni ogen me abolism and showing a wide spec um o se e i y. Because mos pa ien s wi h HE also su e om ch onic li e disease, po o-sys emic shun s, de eloped hype ammonemia and sys emic in lamma ion i is highly desi able ha animal models would include hese ac s (Fig. 1). INDUCED CIRRHOSIS IN RATS BY CARBON TETRACHLORIDE This model is easonably easy o ep oduce, al hough a signi ican numbe o animals die du ing CCl4 ea men , especially i i is main ained un il he onse o asci es. Mul- iple esea ch g oups ha e emphasized, mo eo e , ha a e usually de ec ed li le change in beha io despi e e y ad anced and decompensa ed s a e o li e disease is achie ed. Al hough he well-compensa ed ci hosis model can only be use ul o in es iga e he e ec s o hype am- monemia (HA) (3), which is usually mode a e, i has been shown ha he e is no a se e e educ ion o enzyme ac i i y o he u ea cycle (4), which is commonly ound in pa ien s wi h ci hosis. Fo his eason, Snodg ass (4) s a ed ha he model was inapp op ia e o he s udy o ch onic li e dis- eases. I migh be in e es ing o ep oduce an ad anced s age o ci hosis, bu he p esen a ion o se e e asci es complica es he in e p e a ion o esul s ega ding he ani- mals’ beha io . Thus, cu en ly, CCL4model was no u i- lized o HE esea ch p oposes any mo e. BILE DUCT LIGATION Obs uc ion o he main bile duc induces a seconda y bil- ia y ci hosis, simila o ha o humans, so i appea s in ani- Expe imen al models o hepa ic encephalopa hy Daniel Díaz-Gómez, Ma ía Jo e , José An onio del-Campo, An onio Galindo and Manuel Rome o-Gómez UCM Diges i e Diseases and CIBERehd. Hospi al Uni e si a io de Valme. Uni e sidad de Se illa. Se illa, Spain 1130-0108/2011/103/10/536-541 REVISTA ESPAÑOLA DE ENFERMEDADES DIGESTIVAS Copy igh © 2011 ARÁN EDICIONES, S. L. REV ESP ENFERM DIG (Mad id) Vol. 103. N.° 10, pp. 536-541, 2011 Recei ed: 07-07-11. Accep ed: 08-07-11. Co espondence: Manuel Rome o Gómez. UCM Diges i e Diseases and CIBERehd. Hospi al Uni e si a io de Valme. Uni e sidad de Se illa. A enida de Bella is a, s/n. 41014 Se illa, Spain. e-mail: [email p o ec ed] POINT OF VIEW Díaz-Gómez D, Jo e M, Del-Campo JA, Galindo A, Rome o- Gómez M. Expe imen al models o hepa ic encephalopa hy. Re Esp En e m Dig 2011; 103: 536-541. Vol. 103. N.° 10, 2011 EXPERIMENTAL MODELS FOR HEPATIC ENCEPHALOPATHY 537 REV ESP ENFERM DIG 2011; 103 (10): 536-541 mals a seconda ily li e ailu e, de eloping jaundice, po al hype ension (5), po o-sys emic shun (6), bac e ial anslo- ca ion and immune dys unc ion (7). Bile duc -liga ed a s do no de elop encephalopa hy despi e ammonia le els can be inc eased (8), al hough hese animals ha e shown a loss o spon aneous mo o ac i i y (9) and memo y de ici s (10). Mul iple a emp s we e ca y ou o ind a model using a bil- ia y liga ion based echnique –easy o pe o m– able o de el- op episodes o unambiguous hepa ic encephalopa hy. In his sense, a model was desc ibed in which encephalopa hy was mani es ed a e a po aca al anas omosis in ci ho ic a s by bile duc liga ion, al hough his model was no widely dis ibu ed, p obably because o he high mo ali y o he su gical shun in ci ho ic animals (11). I has ecen ly been p oposed a model ha combines bile duc liga ion wi h a s ed wi h ammonia en iched die , which ha e been ound o inc ease he ammonia le els in he blood and in he b ain (8) bu he p esence o beha io al changes in hese animals, sug- ges ing he de elopmen o encephalopa hy, has no been ound ye . Bile duc obs uc ion p ecludes es ing bilia y- sec e ed d ugs. Ne e heless, BDL model is easy o pe o m and allow us o s udy ammonia me abolism in combina ion wi h an in lamma o y en i onmen . In p elimina y s udies, i has been shown ha he educ ion o ammonia wi h L- o ni hine phenylace a e (OP) is e ec i e in BDL model. Adminis a ion o OP esul s in inc eased con e sion o glu- ama e o glu amine by s imula ion o glu amine syn hase ac i i y in he muscle wi h he subsequen exc e ion o pheny- lace ylglu amine in he u ine, a eac ion in which one mol- ecule o ammonia is emo ed. Also, OP esul ed in no mal- iza ion o glu aminase ac i i y in he gu , indica ing ha OP e ec i ely es ic s he p oduc ion o in i o ammonia in a BDL model (9). These indings sugges de eloping app oach- es o a ge hese enzymes o p e en ammonia elease and HE is a alid he apeu ic s a egy in BDL model. PORTOCAVAL SHUNT IN RATS Many s udies ha e been pe o med in a s wi h po a- ca al shun (PCS) which ha e e ealed some key knowl- edge abou he hype ammonemia in li e diseases (12) (Fig. 2). Ini ially, doub s a ose abou he adequacy o he model o ep oduce he symp oms in animals ha could be equi - alen o human HE, bu acco ding o Bengss on and co- wo ke s (13-15) as well as o he au ho s (16,17), changes in beha io ha e been demons a ed in his model, espe- cially in he a eas o spon aneous ac i i y in esponse o a new en i onmen and explo a o y ac i i y. Mo eo e , i was ound ha hese changes we e e e sible by applying o he animal e ec i e ea men o HE and, as men ioned be o e, he model can be ele an o s udying he HE. Speci ically, Conjee a am and co-wo ke s (18) demon- s a ed ha by adding neomycin o d inking wa e o a s wi h PCS he mo o ac i i y was educed a e exposing hem o a new and da k en i onmen , while Coy and col- leagues also did so using a mo e sophis ica ed echnique by measu ing dis u bances o he ci cadian mo o ac i i y (19). This g oup also demons a ed ha ope a ed a s shown he same imp o emen when hey we e subjec ed o low p o ein die . I should be emembe ed, howe e , ha he me e ac o subjec ing a s o a PCS canno gua an ee he de elopmen o beha io al changes. Aspec s such as he su gical echnique employed o he size o he s oma o he mic o ascula anas omosis, which can ha e a signi ican in luence on he shun p essu e g adien ; o he die p o- ided, he ime om he in e en ion un il he s udy is pe - o med in a s and e en he age and weigh o animals, may a ec he abili y o esea che s o de ec signi ican changes (20,21). Fu he mo e, i should be no ed ha hepa ic neo- ascula iza ion may occu spon aneously de eloping hepa opedal shun s, which a e associa ed wi h eco e y o weigh and dec ease in HAM and sub-clinical HE in hese animals. Lee and Fishe (22) in 1961 and Bismu h in 1962 (23) desc ibed a echnique o di ec po aca al anas omosis, eli- able and ep oducible, p oduced by ascula mic osu u e, which e en oday con inues in o ce by i ue o i s good esul s and ep oducibili y by di e en esea ch g oups. Howe e , because o he echnical di icul y ha is associ- a ed, he e ha e been se e al a emp s o simpli y i u he . Funo ics (24) in 1974 desc ibed a echnique o anas omosis media ed by using a Te lon ube ha was in ended o a oid di ec su u ing and Je kins (21) in 1988, one in which he junc ion be ween he po al and ca a eins was pe o med using cyanoac yla e adhesi e, wi h he in en ion o acili a e handling o mic osu gical essel and he e o e, he ech- nique i sel . Howe e , u he s udies compa ing hese al e - na i e ypes o shun s desc ibed by Bismu h and Lee con- cluded ha he p essu e g adien be ween he po al and ca a sys ems we e lowe a e su gical shun s. This would sugges ha he mic osu ge y anas omosis main ain a a e o long- e m iabili y highe han he al e na i e shun s, which p esen ed highe g adien s because o hei g ea e endency o be in he pos ope a i e pe iod. This would explain he a iabili y o esul s ha had been p e iously desc ibed using hese al e na i e su gical echniques (20). Fig. 1. Physiopa hology o hepa ic encephalopa hy. 538 D. DÍAZ-GÓMEZ ET AL. REV ESP ENFERM DIG (Mad id) REV ESP ENFERM DIG 2011; 103 (10): 536-541 Numa a published in 1983 a modi ica ion o he ech- nique ha g ea ly simpli ies and makes i easie and mo e ep oducible o minimize he ope a i e ime, a key issue in he iabili y o he animal a e he in e en ion (25). This echnique is based on a eno- enous mic osu gical anas- omosis side- o-side be ween he po a and ca a eins o he a , allowing a g ea e calibe o he shun -side-end anas omosis o Lee, Fishe and Bismu h, wi h a heo e i- cally lowe a e o long- e m s enosis. Mo eo e , his ech- nique allows i s use as side- o-side anas omosis, depending on how is le as ollowing comple ion o he su gical shun , o end-side, i i is ied and cu a he p oximal side o he po al ein immedia ely a e making anas omosis, enabling applica ion o expe imen al s udies based on di e en pu - poses (Fig. 3). Since nei he he psychome ic es s no clinical diag- nosis o HE can be achie ed in a s, we need o use al e - na i e me hods. Se e al au ho s ha e de ailed some e lex es s and e alua ing esponses o s imuli (26), di icul o in e p e , o compu e ized ac i i y measu emen s (13,14) o ci cadian hy hms (19), which ep esen a o wa d s ep in his sense. Acco ding o Mullen (27), a s subjec ed o PCS ha e an unce ain esponse o he spon aneous mo o ac i i y, so e en doing an adequa e shun ; a la ge numbe o animals is equi ed o demons a e signi ican beha io al changes in ope a ed animals compa ed o con ols, despi e he use o mo e sophis ica ed me hods o measu emen . Di e en sys ems ha e also been pos ula ed o measu e e oked po en ials (28) o he analysis o encephalog aphic pa e ns (29), wi h low alida ion o da a in HE animal models. Ano he c ucial aspec is he echnique o he shun . A wide su u ed s oma, made wi h low ascula occlusion and an expe su geon a e he basis o a las ing ime po o-sys- emic ci cui and will be able o ep oduce p ope ly he e ec s o HE, compa ed o echniques in which he shun Fig. 2. Fou s eps o po aca al shun in a s. Vol. 103. N.° 10, 2011 EXPERIMENTAL MODELS FOR HEPATIC ENCEPHALOPATHY 539 REV ESP ENFERM DIG 2011; 103 (10): 536-541 is media ed by a “bu on” o made wi h cyanoac yla e, asso- cia ed wi h he e e sibili y o hei e ec s (27). The com- plexi y o s udies o assess encephalopa hy in a s in e ms o hei beha io al changes and lack o s anda diza ion, has led us o limi he alida ion o he model o weigh loss and he s udy o biochemical changes ha ake place in he hepa ic encephalopa hy synd ome. A e iew a icle has been ecen ly published in ela ion o animal models o he s udy o HE (30) ha s ipula es ha PCS in a s would be an app op ia e model o he ype B- EH, whe e he e is a po o-sys emic by-pass wi hou li e ailu e. Howe e , as no ed by Gandhi (31), “ he PCS is a model o li e a ophy o o e 100 yea s”, e e ing o he wo k o Hahn in he nine een h cen u y (32) and cha - ac e ized in g ea de ail in Bismu h’s s udies (33) du ing he 60s o las cen u y. In his ea ly wo k was desc ibed as “ he appea ance o he li e a lapa o omy pe o med a ew days la e o he a s subjec ed p e iously o PCS is pale, and a e sac i icing he animal and weigh i , he e was a signi ican educ ion in i s size.” This loss o li e size is no pa allel o he educ ion in weigh o he a , because he li e -weigh / animal-weigh a io is also educed (34). In he li e unde he mic oscope, a seconda y a ophy is obse ed ha is no accompanied by changes in he com- posi ion in e ms o he basic cellula componen s (wa e , lipids, ca bohyd a es o p o eins) o he hepa ic a chi ec u e. I seems o be ela ed o hepa ocy e a ophy, which begins e y ea ly a e su ge y and may las o se e al mon hs du ing pos ope a i e pe iod (35). Acco ding o hepa ocy e unc ion s udies, se e al changes ha e been demons a ed in hese a s, as a decline in he p oduc ion o bile sal s (abou 40%), a loss o conjuga ion o bili ubin, an al e a ion o he me abolism o bilia y elimina ion o ce ain dyes, such as indocyanine g een –o en used as a diagnos ic me hod o assessing he hepa ic unc ional ese e be o e hepa ec omy in ci ho ic pa ien s (36)–, al e a ions in pla ele agg ega ion and hepa ic d ug me abolism (37). Hemodynamic and ho monal ac o s may be in ol ed in he genesis o his phenomenon. The PCS leads o a loss o blood low h ough he li e , despi e he signi ican inc ease, albei no su icien h ough he hepa ic a e y. The main a gumen s o his hypo hesis de i ed om expe - imen al obse a ions whe e he a ophy is co ec ed almos comple ely a e a po aca al ansposi ion o modi ica ion o he po al ein (38). Mo eo e , he esponsibili y on he phenomenon o loss in subs ances passing h ough he li e wi h hepa o opic e ec can be a gued, mainly wi h pan- c ea ic o igin and ha will a ec li e egene a ion. Among hese, insulin and glucagon ha e been he mos ex ensi ely s udied (39). Bi che ag ees “ he highligh s aking place in his model a e he po o-sys emic shun and loss o unc- ional li e pa enchyma (40), implying ha he model may be conside ed app op ia e o ep esen al e a ions in HE ype C” (which is associa ed wi h LC). Neu on-ana omical lesions ha appea in he encephalopa- hy o li e disease a e simila o hose ound in pa ien s wi h se e e hype ammonemia by gene ic de ec s o he enzymes o he u ea cycle. These indings we e i s desc ibed by Von Hosslin and Alzheime in 1912 (41) and in he case o li e disease, hese lesions a e usually e e sible a e co ec ion o he inc eased le el o ammonia. These cells show a e y pale enla ged nucleus, some imes de o med, wi h a p omi- nen nucleolus and a ma ginal pa e n o ch oma in su ound- ed by a hin cy oplasmic laye , named as Alzheime ype II cells (42). I is no uncommon in hese pa ien s o ind a spongi o m degene a ion o he deepe laye s in he b ain co ex and he basal glia (43). The e a e no s udies on di - e en expe imen al models o HE whe e he neu o-ana om- ical changes a e desc ibed comple ely (44), al hough some au ho s ha e demons a ed he p esence o cha ac e is ic Alzheime cells ype II a e he comple ion o PCS in a s, conside ed as he mos cha ac e is ic neu on-ana omical al e - a ion o HE (45-48). Pilbeam and co-wo ke s published in 1983 (44) a s udy on a b ain subjec ed o PCS and sac i iced a di e en ime poin s a e su ge y ha ques ioned he p esence o Fig. 3. Plasma ammonia le els in simula ed su ge y, po aca al shun ed and bile duc liga ion a s. Table I. Ten leap o pe o m po aca al shun 1. To use inhala ion anes hesia 2. Wide dissec ion o po a and ca a eins 3. A oiding ascula inju y 4. Co ec la e al clamp o bo h eins 5. Clamp ime sho e han 15 minu es 6. S omas should be 5 mm a leas 7. B aided su u e 7/0 wi h cylind ical needles 8. Hemos asis a e declamping by so comp ession 9. Liga ion and po al ein sec ion 10. Isola ion o animals in sepa a e cages o a oid inju ies 540 D. DÍAZ-GÓMEZ ET AL. REV ESP ENFERM DIG (Mad id) REV ESP ENFERM DIG 2011; 103 (10): 536-541 Alzheime ype II as ocy es in he ope a ed animals. They s udied he b ains o only 8 a s (2 animals wi h 2, 4, 16 and 30 weeks a e su ge y). They ound ha he e we e no signi ican di e ences in ammonia plasma alues be ween ope a ed and con ol animals (none o hem he mean alue o plasma AM was e en double ha o he con- ol g oup). In a s udy pe o med by ou g oup whe e 24 a s we e used, 8 and 16 unde PCS in wo con ol g oups o 8 each (con ol and sham), he alues o ammonia plasma o ope a ed a s we e ound 4 o 5- old highe han con ol animals (49). Because o he de ails o he su gical ech- nique, such as he ex en o he s oma be ween he po al and ca a eins, o en ha e impo an esul s ega ding he abili y o ep oduce he hype ammonemia in he li e (20,21), we hough ha pe haps he e we e some p oblems in he su gical echnique, leading o he low le els o plasma ammonia in a s epo ed in he s udy, esponsible o abno - mal b ain his ology. PORTOCAVAL SHUNT WITH ADDITIONAL HANDLING The idea o ep oducing he symp oms o a mo e se e e encephalopa hy in a s (and occasionally in dogs) has been he o igin o he design o di e en manipula ions in he ope a ed animals. A maneu e wi h po en ial u ili y is he adminis a ion o ammonium esins o a s wi h PCS, which appa en ly causes a e e sible encephalopa hy. Ammonium managed by o he means ( o example i. .) clea ly p oduces coma o al, while s ill is no de e mined wha his means in human. Po en ially, his coma is associa ed wi h induced b ain edema, e en hough he e is no e idence on his issue. Fu he mo e, he e is no da a demons a ing he e e sibili y o he coma. No mal a s which we e ed wi h ammonium ace a e de eloped hype ammonemia wi h a simila deg ee o a s wi h PCS, and ye hey we e esis an o he ime- poin adminis a ion o addi ional ammonia, bu he a s wi h PCS we e, as men ioned abo e, in he opposi e si u- a ion (50). The supe posi ion o an acu e oxic hepa i is wi h dime hyl-ni osamide in dogs wi h PCD has been shown o induce a se e e HE (51). Al e na i e ways o induce HE on animals wi h PCS included blood ex ac ion o he in ake o high amoun s o p o ein in he die , which was e ec i e in dogs, bu did no in a s. Pa ial hepa ec omy has also been used in some cases in PCS a s, o PCS o a s wi h ci hosis induced by e achlo ide ca bon, bu changes in animal beha io we e no epo ed. Di e en wo ks ha e been ecen ly published using a comple e bilia y liga ion in a s wi h p e ious PCS, which induced an acu e o subacu e choles asis (11). This model has no been ully cha ac e ized and has he added di icul y o inducing a se e e weigh loss, p obably caused by ano - exia o malabso p ion. In summa y, he ideal HE animal model emains elusi e. Po aca al shun ed a emains as he gold s anda d me hod o HE in animals. Easie o pe o m models like bile duc liga ion could be use ul as complimen a y model. The use- ulness o adding me hods equi e u he s udies. Po a- ca al shun in CCL4-induced ci ho ic a s could be in igu- ing bu ex emely di icul wi h highe a mo ali y. Hype ammonia-based die o hiace amide-induced li e ailu e could also be aken in mind when de eloping com- bina ion models. Po aca al shun is a good model o min- imal hepa ic encephalopa hy and could be use ul o he de elopmen o new he apeu ic agen s. REFERENCES 1. Mullen KD, McCullough AJ. P oblems wi h animal models o ch onic li e disease: sugges ions o imp o emen in s anda diza ion. Hepa- ology 1989;9(3):500-3. 2. 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