Are in vitro and in silico approaches used appropriately for animal-based major depressive disorder research?

RESEARCH ARTICLE A e in i o and in silico app oaches used app op ia ely o animal-based majo dep essi e diso de esea ch? Cons anc¸a Ca alhoID 1 *, Susana A. M. Va ela 2¤ , Tiago A. Ma ques 3,4 , And ew Knigh 5 , Luı ´s Vicen e 1 1Faculdade de Ciências, Cen o de Filoso ia das Ciências, Uni e sidade de Lisboa, Lisboa, Po ugal, 2Faculdade de Ciências, cE3c – Cen e o Ecology, E olu ion and En i onmen al Changes, Uni e sidade de Lisboa, Lisboa, Po ugal, 3Cen e o Resea ch in o Ecological and En i onmen al Modelling, Uni e si y o S And ews, S And ews, Sco land, 4Depa amen o de Biologia Animal, Faculdade de Ciências, Cen o de Es a ı ´s ica e Aplicac¸ões, Uni e sidade de Lisboa, Lisboa, Po ugal, 5Cen e o Animal Wel a e, Uni e si y o Winches e , Winches e , Uni ed Kingdom ¤Cu en add ess: Ins i u o Gulbenkian de Ciência, Oei as, Po ugal *cons anca.ca [email protected] Abs ac The cu en pa adigm o biomedical esea ch and d ug es ing pos ula es ha in i o and in silico da a in o m animal s udies ha will subsequen ly in o m human s udies. Recen e i- dence poin s ou ha animal s udies ha e made a poo con ibu ion o cu en knowledge o Majo Dep essi e Diso de , whe eas he con ibu ion o in i o and in silico s udies o animal s udies- wi hin his esea ch a ea- is ye o be p ope ly quan i ied. This quan i ica ion is impo an since biomedical esea ch and d ug disco e y and de elopmen includes wo s eps o knowledge ans e abili y and we need o e alua e he e ec i eness o bo h in o de o p ope ly implemen 3R p inciples (Replacemen , Reduc ion and Re inemen ). He e, we used he ci a ion acking acili y wi hin Web o Science o loca e ci a ions o o iginal esea ch pape s on in i o and in silico ela ed o MDD published iden i ied in PubMed by ele an sea ch e ms. 67 publica ions desc ibing a ge pape s we e loca ed. Bo h in i o and in silico pape s a e mo e ci ed by human medical pape s han by animal pape s. The esul s sugges ha , a leas conce ning MDD esea ch, he cu en wo s eps o knowledge ans e abili y a e no being ollowed, indica ing a poo compliance wi h he 3R p inciples. 1. In oduc ion Biomedical esea ch hea ily elies on animal s udies, despi e he e hical and clinical limi a ions o hese [1]. The s anda d con empo a y pa adigm o biomedical esea ch, and d ug disco e y and de elopmen , equi es scien is s o es pu a i e new clinical in e en ions, by p og essing om simple o inc easingly complex models, p io o conduc ing human s udies and ials, as shown in Fig 1. PLOS ONE PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0233954 June 24, 2020 1 / 6 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Ci a ion: Ca alho C, Va ela SAM, Ma ques TA, Knigh A, Vicen e L (2020) A e in i o and in silico app oaches used app op ia ely o animal-based majo dep essi e diso de esea ch? PLoS ONE 15 (6): e0233954. h ps://doi.o g/10.1371/jou nal. pone.0233954 Edi o : Jyo shna Kanungo, Na ional Cen e o Toxicological Resea ch, UNITED STATES Recei ed: Feb ua y 28, 2020 Accep ed: May 11, 2020 Published: June 24, 2020 Copy igh : ©2020 Ca alho e al. This is an open access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Da a A ailabili y S a emen : All analysed pape s a e a ailable a h ps://jou nals.sagepub.com/doi/ pd /10.1177/0261192919885578. Funding: This s udy was suppo ed by Animal ee Resea ch–Swi ze land in he o m o unds awa ded o CC. Cen o de Es a ı ´s ica e Aplicac¸ões (CEAUL) p o ided pa ial suppo o TAM, h ough p ojec unding om Fundac¸ão pa a a Ciência e a Tecnologia, Po ugal (FCT) (UID/MAT/00006/ 2019). The publica ion ee was inanced by Po uguese na ional unds awa ded o LV wi hin E en hough his pa adigm is mo e ocused owa d d ug disco e y, i is also encou aged o b oade esea ch, by legisla ion and guidelines pe aining o animal esea ch, in a ious coun- ies and egions (e.g.[3]). Suppo e s o animal s udies wi hin biomedical esea ch claim ha 1) i is no possible o discon inue hei use, as ha would jeopa dize human heal h, and ha 2) human-based me h- ods (in silico and in i o) a e used in ea ly s eps o biomedical esea ch o in o m he animal esea ch communi y, hence a oiding unnecessa y o excessi e use o animals. Fo example, pu po edly, i a subs ance shows high le els o oxici y in i o i will no p og ess in o animal es ing [4]. In he same way, a d ug ha shows high oxici y in animal es ing should no p o- ceed o human ials. Howe e , i has been demons a ed ha human ials may some imes occu simul aneously wi h animal ials, a he han sequen ially, as one would expec i animal ials we e an essen ial s ep p io o human ials [5]. In ou p e ious s udy we compa ed he numbe o ci a ions in i o,in silico and non- human p ima e-based (NHP) o iginal s udies ocused on Majo Dep essi e Diso de (MDD), ha we e ecei ed (i) in o al, (ii) by unspeci ied human medical pape s, and (iii) by human medical pape s ocused on MDD. We e i ied ha bo h in i o and in silico esea ch pape s ecei ed mo e ci a ions by human medical pape s, han NHP pape s. This was unexpec ed, conside ing ha mos coun ies es ic he use o NHPs, making i easonable o p esume ha when hey we e used, hey should p o ide a signi ican con ibu ion o human heal h. How- e e , his was no he case. Da a ob ained ia simple models (in i o and in silico) seemed o be mo e isible o conside ed mo e impo an by he human medical esea ch communi y. This called in o ques ion he con empo a y pa adigm o biomedical esea ch and d ug disco - e y, in which knowledge is p esumed o ans e be ween animal and human models [6]. Conside ing ha his pa adigm p esumes wo s eps o knowledge ans e abili y: i) be ween simple and complex models, and ii) be ween animals models and humans, we wonde ed i he e could be knowledge ans e abili y p oblems in s ep (i), simila o hose we demons a ed a s ep (ii). Hence, he aim o he cu en s udy is o assess whe he in i o and in silico pape s desc ib- ing o iginal da a on a human diso de (MDD) a e being app op ia ely ci ed by subsequen ani- mal-based pape s. I is impo an o men ion ha animal models a e ex ensi ely used in MDD esea ch. In ac , by he ime ou s udy was conduc ed he e we e abou wice as many o iginal pape s using animal models in MDD esea ch han pape s using in i o and in silico app oaches. Du ing s udies ocused on MDD, animals equen ly unde go se e e p ocedu es such as lea ned helplessness o o ced swim es p o ocols. Mos applicable legisla ions and guidelines manda e ha such p ocedu es should be a oided whe e e possible. Hence i is easonable o expec ha he MDD- ocused animal esea ch communi y should be pa icula ly ale o he da a and insigh s p o ided by simple da a. E en hough he e is a wide consensus ha he use o simple models such as in i o and in silico me hods wi hin basic and applied biomedical esea ch helps animal esea che s o mee he p inciples o Replacemen (o animals wi h al e na i es) and Reduc ion (o animal num- be s), as desc ibed by Russell & Bu ch [7], o ou knowledge, he e has ne e been a sys ema ic Fig 1. Cu en pa adigm o biomedical esea ch and d ug disco e y and de elopmen . Kindly p o ided by Taylo [2]. h ps://doi.o g/10.1371/jou nal.pone.0233954.g001 PLOS ONE In i o and in silico use in animal-based majo dep essi e diso de esea ch PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0233954 June 24, 2020 2 / 6 he Cen o de Filoso ia das Ciência da Uni e sidade de Lisboa (CFCUL) esea ch cen e s a egic p ojec , unded by Fundac¸ão pa a a Ciência e a Tecnologia (FCT) (UIDB/00678/2020). These unde s played no ole in s udy concep ion, design, da a collec ion and analysis, manusc ip au ho ship o he decision o submi o publica ion. Compe ing in e es s: No au ho s ha e compe ing in e es s. s udy ha empi ically e i ies whe he animal esea che s a e, indeed, applying his p inciples o hei p ac ice i.e. i hey a e loca ing and using applicable da a ob ained ia such simple models. I in i o and in silico s udies a e indeed seen as an impo an s ep p io o conduc ing ani- mal s udies in biomedical esea ch, and animal s udies a e in u n seen as impo an p io o conduc ing human s udies, hen we would expec ha pape s desc ibing in i o o in silico da a on a human diso de should be ci ed mo e equen ly by animal pape s, han by human medical pape s. I , on he con a y, his is no he case, hen u he s udies on o he human diso de s and d ug de elopmen should be conduc ed o con i m he ex en o which he con- empo a y heo e ical pa adigm o biomedical esea ch is ac ually being ollowed in p ac ice. I adhe ence is no as common as belie ed, hen his pa adigm should clea ly be e ised. 2. Me hods We conduc ed a ci a ion analysis as de ined by Ga ield and Me on [8]. Concisely, in a ci a- ion analysis, a ge pape s a e loca ed i s and hen a sea ch o all o he pape s ci ing he o - me is pe o med. The in o ma ion compiled comp ises he o al numbe o ci a ions, and he pa e ns o ci a- ion. We used a o al o 67 a ge pape s o in i o o in silico s udies on MDD- u ilising only human da a, selec ed om he ci a ion analysis da abase c ea ed in ou p e ious s udy [6]. The ci a ion analysis was pe o med be ween Sep embe 2016 and June 2017. We conside ed all published pape s using in i o o in silico me hods, ha aimed o gain knowledge abou MDD, and we e published p io o 2011, o enable i e-yea ime o ci a ions–a equen ly used imeline o ci a ion analysis [9]. To loca e a ge pape s we sea ched PubMed– he la ges eely accessible bibliog aphic da abase, using he ollowing Medical Subjec Heading (MeSH) sea ch e ms: ‘Dep essi e Diso de , Majo ’ AND (“in silico” OR ‘compu e model’ OR ‘ma he- ma ical model’ OR ‘compu e simula ion’ OR ‘in i o’ OR ‘cell cul u e’ OR ‘cul u e echnique’ OR ‘cell line’ OR ‘o gan cul u e’ OR ‘ issue cul u e’. Ou goal was o selec o iginal publica ions ha p esen ed new da a, so we used PubMed il e s o exclude e iew a icles (“ e iew”, “sys- ema ic e iew”, “me a-analysis”, “bibliog aphy”) as well as opinion a icles (“biog aphy”, “au obiog aphy”, “commen ”, “edi o ial”, “in e iew”). We also excluded by hand in i o pape s ha used animal issue o cells. Using he ci a ion acking acili y wi hin Web o Sci- ence, we coun ed he numbe o imes each a ge pape was ci ed by subsequen pape s in he ollowing ca ego ies: ‘animal esea ch pape s‘, ‘human medical pape s‘, ‘in i o pape s‘, and ‘in silico pape s‘. Ci ing pape s may ha e been assigned o mo e han one ca ego y i hey desc ibed di e en esea ch app oaches (e.g. human-based and in i o). 3. Resul s In o al, 464 (18%) o he 2,574 ci a ions ecei ed by he 38 in i o pape s we e by in asi e ani- mal esea ch pape s, and 978 (40%) we e by human medical pape s. Fo he 29 in silico pape s, 44 (5%) o he 806 ci a ions we e by in asi e animal esea ch pape s, and 317 (39%) by human medical pape s. As shown in Fig 2, he majo i y o ci a ions ecei ed by bo h in i o o in silico a ge pape s we e by pape s employing he same esea ch me hod, and by human medical pape s. The p o- po ion o ci a ions by animal pape s and he o he esea ch me hod we e conside ably lowe . Mo e impo an ly, he p opo ion o ci a ions by animal pape s was lowe han by human medical pape s. PLOS ONE In i o and in silico use in animal-based majo dep essi e diso de esea ch PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0233954 June 24, 2020 3 / 6 4. Discussion The esul s o ou ci a ion analysis sugges ha he s anda d app oach o es ing medical hypo heses–which pos ula es ha in i o and in silico esea ch is an impo an s ep p io o conduc ing animal es ing–is no suppo ed by ci a ion da a, a leas o MDD esea ch. Clea ly, MDD biomedical esea ch u ilising in i o and in silico da a does no seem o be con- side ed impo an by, o a leas mo e impo an o, he animal esea ch communi y, han i is o he human medical communi y. One can a gue ha i he animal esea ch communi y is no ci ing in i o and in silico pape s on MDD, hese migh be o limi ed use. Howe e , ha is inconsis en wi h hei sub- s an ial use by he human medical communi y, which ci es mo e his kind o esea ch han esea ch based on animal s udies [6]. Addi ionally, his lack o ans e abili y o knowledge be ween he animal and he human medical esea ch communi ies is u he e idenced by he ac ha , in gene al, mos ci a ions ecei ed by animal esea ch pape s a e wi hin o he ani- mal-based s udies, a he han wi hin human medical pape s [10]. MDD is a complex human men al diso de wi h mul i ac o ial ae iopa hogenesis [11], so one canno ex apola e ha he ci a ion pa e ns ound he e will necessa ily be eplica ed in o he diso de s ha ha e jus one cause (e.g. Down’s synd ome). Fu he mo e, a single disease analysis is no enough o gene alize he esul s o he en i e ield o biomedical esea ch. Hence, he nex s ep should be he use o a simila app oach a ge ing mono ac o ial diso - de s and d ug ials. I , as whole, hese s udies p oduce simila esul s, hen i would be com- pelling e idence ha he accep ed pa adigm o biomedical esea ch and d ug disco e y and Fig 2. Boxplo s o he p opo ion o ci a ions ecei ed by esea ch ca ego y o in i o A) and in silico B) pape s on MDD. h ps://doi.o g/10.1371/jou nal.pone.0233954.g002 PLOS ONE In i o and in silico use in animal-based majo dep essi e diso de esea ch PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0233954 June 24, 2020 4 / 6 de elopmen is no being su icien ly ollowed, which suppo s he claims made by se e al au ho s [1] ha he 3Rs a e no being add essed as well as equi ed by applicable legisla ion and good esea ch p ac ice. This sugges s ha animal s udies in biomedical esea ch a e mos ly de ining hei esea ch p io i ies au onomously, a he han being pe ec ly amed in he bio- medical esea ch pa adigm. Six y yea s ago Russell and Bu ch [7] es ablished he ounda ions o much cu en legisla- ion ega ding animal expe imen a ion, wi h he o mula ion o he 3R p inciples. E en hough he esea ch communi y unanimously welcomes hem, he ocus o hei applica ion has p edominan ly been e inemen , and no always in an e ec i e way [1]. Nowadays he e is an inc easing numbe o da abases on human and animal p o ein exp ession di e ences ( o a e iew see [12]) which, on he one hand, makes i easie o esea che s o loca e and ci e exis ing da a; bu , on he o he hand, migh s imula e animal esea ch o be conduc ed independen ly o in i o and in silico da a o popula e such da abases. In heo y, he educ ion p inciple depends upon he s anda d use o in silico and in i o echniques p io o animal s udies. I o iginal da a on human diso de s om in i o and in sil- ico app oaches a e no being used by he animal esea ch communi y, hen he educ ion p in- ciple is no being p ope ly ul illed. The easons behind his mus su ely be mul iple. One o he possible easons is he inadequacy o sys ema ic e iews ha animal esea che s some imes pe o m on hei esea ch opic, p io o conduc ing animal expe imen s. These should p e en unnecessa y animal use [13], bu by excluding om he sea ch in i o and in silico s udies, esea che s can exclude an impo an sou ce o knowledge. Based on ou esul s we ecommend ha changes a e made in cu en sys ema ic e iew p o ocols in o de o include in i o and in silico da a. Ano he eason ha became salien wi h ou s udy and dese es a en ion, is ha in i o and in silico app oaches a e, by de ini ion, human-based me hods, no animal-based me hods. Concei ably human da a is no ele an enough o animal pape s, in he same way animal s udies do no seem o be ele an o subsequen human s udies [6,10]. This highligh s ha he cu en pa adigm o biomedical esea ch and d ug disco e y and de elopmen includes wo s eps o knowledge ans e abili y be ween he animal and he human models, nei he o which appea o wo k well. I simila esul s a e ound in o he dis- o de s and mo e impo an ly, in d ug disco e y, han he cu en pa adigm mus be changed. Speci ically, animal es ing mus be dep io i ized, wi h g ea e in es men in human-based in i o and in silico esea ch app oaches. Suppo ing in o ma ion S1 File. (ODS) Au ho Con ibu ions Concep ualiza ion: Cons anc¸a Ca alho, Susana A. M. Va ela. Da a cu a ion: Cons anc¸a Ca alho, Tiago A. Ma ques. Fo mal analysis: Cons anc¸a Ca alho, Tiago A. Ma ques. In es iga ion: Cons anc¸a Ca alho. Me hodology: Cons anc¸a Ca alho, And ew Knigh . PLOS ONE In i o and in silico use in animal-based majo dep essi e diso de esea ch PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0233954 June 24, 2020 5 / 6 Supe ision: Tiago A. Ma ques, And ew Knigh , Luı ´s Vicen e. Valida ion: And ew Knigh . W i ing – o iginal d a : Cons anc¸a Ca alho, Susana A. M. Va ela. W i ing – e iew & edi ing: Cons anc¸a Ca alho, Susana A. M. Va ela, Tiago A. Ma ques, And ew Knigh . Re e ences 1. He mann K. Re inemen on he way owa ds eplacemen : A e we doing wha we can? In He man K, Jane K, edi o s. 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Labo a o y Animals. 2012; 46(1): 24–31. h ps:// doi.o g/10.1258/la.2011.011087 PMID: 22037056 PLOS ONE In i o and in silico use in animal-based majo dep essi e diso de esea ch PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0233954 June 24, 2020 6 / 6