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Volume 52 Number 1 February 2018 ISSN 0023-6772

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Laboratory Animals

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Volume 52 Number 1 February 2018

Contents

Original Articles

Ultrasonographic and histological evaluation of the effects of long-term carotid catheterization oncardiac function in NMRI mice 17Anne C Teilmann, Morten B Thomsen, Elizabeth A Ihms, Nathan Pate, Jann Hau and Klas Abelson

Diurnal rhythms of blood glucose, serum ghrelin, faecal IgA and faecal corticosterone in ratssubjected to restricted feeding using the diet board 29Iiris Kasanen, Katja Inhila, Eriika Savontaus, Hanna-Marja Voipio, Kai Okva, Satu Mering, Vesa Kiviniemi, Jann Hau,

Sakari Laaksonen and Timo Nevalainen

Diagnostic imaging modalities and surgical anatomy of the temporomandibular joint in rabbits 38Michal Kyllar, Barbora Putnova, Vladimır Jekl, Ladislav Stehlık, Marcela Buchtova and Jan Stembırek

Sedative and cardiorespiratory effects of detomidine constant rate infusion in sheep 51Rauane Sousa de Moura, Isabela Plazza Bittar, Luiz Henrique da Silva, Ana Carolina Vasquez Villela, Marcelo Borges

dos Santos Junior, Naida Cristina Borges and Leandro Guimaraes Franco

TNFa depleting therapy improves fertility and animal welfare in TNFa-driven transgenic models ofpolyarthritis when administered in their routine breeding 59Amy J Naylor, Guillaume Desanti, Atif N Saghir and Rowan S Hardy

Laboratory animal science course in Switzerland: participants’ points of view and implications fororganizers 69Fabienne Crettaz von Roten

Quality of reporting of otorhinolaryngology articles using animal models with the ARRIVEstatement 79Aren Bezdjian, Sjaak F L Klis, Jeroen P M Peters, Wilko Grolman and Inge Stegeman

Short Report

Ultrasound guided percutaneous common carotid artery access in piglets for intracoronary stemcell infusion 88Jason H Anderson, Nathaniel W Taggart, Sarah L Edgerton, Susana Cantero Peral, Kimberly A Holst and Frank Cetta

Case Report

Preputial diverticulum dilation in a Goettingen minipig 93Nora Denk, Claudia Senn, Bjoern Jacobsen and Tobias Schnitzer

News

Reorganization of the Journal’s Editorial Board to Improve the Journal’s Productivity and Efficiency 101Beat M Riederer, Paulin Jirkof and Arieh Bomzon

Obituary

Professor Klaus Gartner, 1927–2017 105

Calendar of events/Index to advertisers 108

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Laboratory Animals

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(temporary), S WellsSurgical Procedures D Bouard, R TolbaSystematic Review M Ritskes-Hoitinga,

BS KousholtToxicology F RuttenVeterinary Medicine E Rivera, J Sanchez-Morgado,

L Whitfield,N Kostomitsopoulos

Nutrition and Diets G Tobin

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AFSTALAssociation Francaise des Sciences etTechniques de I’Animal de LaboratoirePresidentSebastian Paturance

Vice PresidentElodie Bouchoux

Secretariat: 28, rue Saint Dominique, 75007,Paris, France(www.afstal.com)

ECLAMEuropean College of Laboratory AnimalMedicinePresidentRony Kalman

Secretariat: PO Box 9101NL-6500HB Nijmegen, The Netherlands(http://eslav-eclam.org/eclam)

ESLAVEuropean Society of Laboratory AnimalVeterinariansPresidentJose Manuel Sanchez-Morgado

Honorary SecretaryMassimiliano Bardotti

Secretariat: Sanofi-Aventis R&D,1, Avenue Pierre Brossolette91385 Chilly-Mazarin Cedex France([email protected]; www.eslav-eclam.org)

FELASAFederation of EuropeanLaboratory Animal Science AssociationsPresidentHanna-Marja Voipio

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Secretariat: PO Box 951,Needham MarketIpswich IP6 8WH, UK(www.felasa.eu)

GV-SOLASGesellschaft fur Versuchstierkunde(Society for Laboratory Animal Science)

PresidentRene Tolba

SecretaryNicole LinklaterFaculty of BiologyPhilipps UniversityKarl-von-Frisch Str. 835043 MarburgGermany(www.gv-solas.de)

ILAFIsraeli Laboratory Animal ForumPresidentAmir Rosner

SecretaryDavid CastelNeufeld Cardiac ResearchInstituteSheba Medical CenterTel Hashomer 52621Israel(www.ilaf.org.il)

LASALaboratory Animal Science AssociationPresidentDavid Anderson

Secretary GeneralMiles Maxwell

PO Box 524, Hull,HU9 9HE, UK(www.lasa.co.uk)

NVPNederlandse Vereniging voorProefdierkunde(Dutch Association for Laboratory AnimalScience)

PresidentMartje Fentener van Vlissingen

SecretaryJan LangemansBPRCLange Kleiweg 1392288 GJ RijswikThe Netherlands(www.proefdierkunde.nl)

SECALSociedad Espanola para las Ciencias delAnimal de Laboratorio(Spanish Society for Laboratory AnimalScience)

PresidentAntonio Martınez Escandell

Vice PresidentMarıa Teresa Rodrıgo Calduch

SecretaryAngel Naranjo Pino

TreasurerCarlota Largo Aramburu

Secretariat: c/Maestro Ripoll, 8,28006 Madrid,Spain(www.secal.es)

SGVSchweizerische Gesellschaft furVersuchstierkundeSociete Suisse pour la Science des Animaux deLaboratoire (Swiss Laboratory Animal ScienceAssociation)

PresidentDr. Birgit Ledermann

SecretaryDr Brunhilde Illgen-WilckeMicroBios GmbHMicrobiological ServiceChristoph-Merian-Ring 31ACH-4153 Reinach, Switzerland(www.scienzenaturali.ch)

SPCALSociedade Portuguesa de Cienciasem Animais de Laboratorio(Portuguese Society for Laboratory AnimalScience)

PresidentIsabel Vitoria Figueiredo

Vice-PresidentRicardo Afonso

SecretaryCatarina Pinto Reis

Secretariat: Laboratorio deFarmacologiaFaculdade de FarmaciaLargo de D. Dinis3000 CoimbraPortugal(www.spcal.pt)

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Original Article

Ultrasonographic and histologicalevaluation of the effects of long-termcarotid catheterization on cardiacfunction in NMRI mice

Anne C Teilmann1, Morten B Thomsen2, Elizabeth A Ihms3,Nathan Pate3, Jann Hau1 and Klas Abelson1

AbstractCatheterization of laboratory mice is commonly performed in biomedical research to infuse substances andfor blood sampling. One approach is to catheterize the right common carotid artery and advance the catheteruntil the tip is positioned in the aorta or the proximal brachiocephalic trunk. Owing to the small body size ofthe mouse, a catheter tends to occupy a great part of even the larger vessel lumens, and this may increasevascular resistance with potential pathophysiological impacts on the heart. The present study comparedcardiac function of catheterized mice, with catheter tip placement in the brachiocephalic trunk, with sham-operated mice and non-operated control mice. During four weeks post-catheterization, M-mode echocardi-ography measurements of the thickness of the left ventricular anterior wall, left ventricular inner diameterand the thickness of the left ventricular posterior wall were performed. The left ventricular volume, ejectionfraction and fractional shortening were calculated. Moreover, aortic recordings of the thickness of the medialand lateral walls as well as the inner diameter were measured. Terminally, histological analysis of the heartswas conducted, and body weights and heart weights were compared between groups. No effects on echo-cardiography parameters, histology, body weights or cardiac weights could be found between groups. In thepresent study, implantation of a carotid catheter with catheter tip placement in the proximal brachiocephalictrunk had minimal influence on cardiac and aortic physiology and did not induce significant cardiac changes.

Keywordsrefinement, echocardiography, mice, catheterization, histology

Date received: 6 December 2016; accepted: 2 April 2017

Vascular catheterization of laboratory mice is frequentlyused for the infusion of substances and for blood sam-pling. Catheterization of the right common carotidartery, where the catheter tip is advanced towards theaortic arch1–3 is a common method. Positioning thecatheter in a major vessel is good surgical practice, asit improves blood flow around the catheter and minim-izes endothelial damage. The benefits, compared withcatheterizing smaller vessels, are improved catheterpatency and a reduced risk of complications such asthrombophlebitis and thromboembolism.4

We routinely catheterize mice in our laboratory andhave previously demonstrated minimal surgical stressand good recovery with this technique.5,6 However,histological data from an unrelated study have revealedsigns of myocardial degeneration of the carotid artery

only four days post-catheterization.7 Increased aorticturbulence may increase cardiac work load and resultin a negative impact on cardiac function.8 This risk

1Department of Experimental Medicine, Faculty of Health andMedical Sciences, University of Copenhagen, Copenhagen,Denmark2Department of Biomedical Sciences, Faculty of Health andMedical Sciences, University of Copenhagen, Copenhagen,Denmark3Department of Molecular and Comparative Pathobiology, JohnsHopkins University, Baltimore, Maryland, USA

Corresponding author:Anne Charlotte Teilmann, Department of Experimental Medicine,University of Copenhagen, Blegdamsvej 3b, Copenhagen 2200,Denmark.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 17–28

! The Author(s) 2017

Reprints and permissions:

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DOI: 10.1177/0023677217706724


increases over time with a sustained increase in cardiacwork load.9 As vascular catheters are frequentlyintended for long-term usage, the potential impacton cardiac function from altered hemodynamics maythus increase with the length of the study period. Weconsidered whether placement of the catheter tip closeto the aortic arch would affect cardiac functionadversely due to a disruption of aortic hemodynamics,as seen in other aortic pathologies such as aortic sten-osis10 and aortic regurgitation,11 and whether long-term carotid catheterization would result in hyper-trophic cardiomyopathy with cardiac dysfunction inthese mice.

To our knowledge, no studies have investigatedcardiac function in mice with a carotid catheter. A thor-ough assessment of the impact of carotid catheteriza-tion on cardiac function in mice is particularlyimportant, as even subclinical pathological heart con-ditions may affect animal welfare and the experimentaloutcome.

The present study investigated cardiac function inmice with a carotid catheter through echocardiographyfor four weeks post-catheterization. Morphometric andfunctional parameters were compared with sham-operated mice and non-operated control mice. Bodyweights (BWs) and cardiac weights were comparedbetween groups, and histopathological analyses of thehearts were conducted to assess tissue level effects fromthe catheterization. The hypothesis was that catheter-ized mice would demonstrate impaired cardiac functionon echocardiography, and present with histological evi-dence of cardiomyopathy in contrast to sham-operatedand control mice.

Animals and housing

The experiments were approved by the AnimalExperiments Inspectorate under the Danish Ministryof Food, Agriculture and Fisheries (license number:2014-15-2934-01055) and by the local animal welfarebody. All animals were handled by trained personnelin accordance with the Guide for the care and use oflaboratory animals12 in an Association for Assessmentand Accreditation of Laboratory Animal Care(AAALAC)-accredited facility.

Twenty-one male BomTac:NMRI mice were pur-chased from Taconic, Ry, Denmark. The animalswere 7–8 weeks old and were acclimatized for oneweek before starting the experiments. The specifiedgender, stock and number of mice were used to testthe hypothesis, based on histological analyses and prac-tical experience from previous catheterization stu-dies.6,7 We generally use outbred NMRI stock in ourcatheterization studies, as these are commonly used ingeneral biological research.13 Furthermore, they are

genetically uniform in comparison to the widevariability of a species but without lacking geneticdiversity, as do the genetically defined inbred mousestrains.14

The mice were randomly distributed into threegroups: one control group (CONTROL, n¼ 7), onegroup in which a catheter was surgically implanted inthe common carotid artery (CATH, n¼ 7) and onesham-operated group, in which the carotid artery wasligated but not catheterized (SHAM, n¼ 7). One cathe-terized mouse did not recover well and was euthanizedas it had reached a humane endpoint, leaving six micein the CATH group.

The mice were housed in Eurostandard type II cages(Tecniplast, Buguggiate, Italy) in an individually venti-lated cage (IVC) system, with aspen chips (Tapvei Oy,Kortteinen, Finland) as bedding. To avoid conspecificinterference with the catheters, all mice were singlyhoused from the day of arrival. Bite bricks (TapveiOy), cardboard tubes (Lillico, Horley, UK) and shelters(JAKO Shelter, Molytex, Glostrup, Denmark) wereused as environmental enrichment. Wood wool(Tapvei Oy) and Nestlets (LBS Biotechnology,Horley, UK) were used as nesting material. Feed(Altromin 1314; Altromin GmbH & Co. KG, ImSeelenkamp, Germany) and acidified tap water wereprovided ad libitum. A diurnal rhythm was maintainedthrough a 12:12 h light–dark cycle starting at 06:00 h,and with a 30min twilight before lights off or on. Cagetemperature was kept at 22�C (�2�C), with a relativehumidity of between 45 and 65%, and approximately75 air changes per hour. The facility followed theFederation for European Laboratory Animal ScienceAssociations (FELASA) guidelines for health monitor-ing in rodent facilities,15 where sentinel mice had testedpositive for Helicobacter spp. but none of the otherpathogens on the FELASA list.

Materials and methods

Surgery

In the second week of the study (Table 1), the mice inthe CATH group had an arterial catheter (MAC-13;SAI Infusion Technologies, Lake Villa, IL, USA) sur-gically implanted in their right common carotid artery,as previously described.6 Anesthesia was induced in aninduction chamber (AgnTho’s AB, Lidingo, Sweden)with 5% isoflurane (Forene�; Abbot Scandinavia,Stockholm, Sweden) delivered in 100% oxygen, usingan anesthetic unit that allowed for constant monitoringand calibration of isoflurane concentration and air flow(U-410 anesthetic unit; AgnTho’s AB). During surgery,the anesthesia was maintained at 2.5–3.0% isoflurane inoxygen, provided through an anesthetic face mask

18 Laboratory Animals 52(1)

(AgnTho’s AB). A midline incision was made in theventral neck, and the right common carotid arterywas isolated by blunt dissection. Two 6-0 vicryl sutures(Ethicon, St-Stevens-Woluwe, Belgium) were placedaround the artery. The rostral suture was completelytied before a small cut was made in the artery betweenthe sutures, and the catheter was inserted. The catheterwas equipped with a retention bead one cm from the tipfor its secure placement inside the vessel and for itscorrect placement of the tip in the proximal brachioce-phalic artery. The properties of the catheter have beendescribed previously.16 The catheter was advanced untilthe retention bead reached the cut surface, and thecaudal suture was cross-tied around the bead. The cath-eter was tunneled subcutaneously to the midscapularregion in the neck and the skin was closed in twolayers. Catheter patency was confirmed by the aspir-ation of blood, which was returned to the animal byflushing with 50 mL of heparinized saline (25 IU/mL),before locking the catheter with 20 mL of heparin:gly-cerol locking solution (Cath-LocHGS; SAI InfusionTechnologies) and a steel plug (SAI InfusionTechnologies). The catheter was hereafter not accesseduntil the end of study.

The sham-operated mice were prepared for surgeryin a similar manner to the catheterized mice. Using thesame approach, the right common carotid artery wascarefully dissected and ligated with a single suture with-out implantation of a catheter. The skin was sutured inthe same manner as described above.

All surgeries were completed before noon and per-formed under aseptic conditions. The mice were givenpre-emptive analgesia by means of 1mg/kg BW bupre-norphine (Temgesic; Schering-Plough, Brussels,Belgium) mixed in a nut paste (Nutella�; Ferrero,Pino Torinese, Italy) one hour prior to surgery andthen once daily for two days post-surgery, as described

previously.17,18 To ensure adequate analgesia on theday of surgery the mice were injected with 0.1mgbuprenorphine/kg BW subcutaneously in the flankbefore terminating anesthesia.

After surgery, the mice were allowed to recover in aquiet room on a heating pad set at 28�C. The well-beingof all mice was monitored through daily inspectionsand recordings of food intake and BWs (data notshown) from three days prior to surgery to three dayspost-surgery, and then weekly throughout theexperiment.

Echocardiography

In weeks 1, 4 and 6 of the study, all mice were subjectedto echocardiography (Vevo 770, VisualSonics�;VisualSonics BV, Amsterdam, The Netherlands)under isoflurane anesthesia. To avoid biased meas-urements from unequal depths of anesthesia, theisoflurane concentration was set such that restingheart rates varied between 300 and 400 beats perminute, which corresponded to 2.5–3.5% of isofluranedelivered in 100% oxygen. During echocardiography,heart rate and respiration were monitored automatic-ally by the software and body temperature with a rectalthermometer (ThermoWorks, American Fork, UT,USA).

Three M-mode measurements were obtained fromthe heart, two in a longitudinal view and one in across sectional view (Figure 1), to calculate an averageapproximate thickness of the left ventricular anteriorwall (LVAW), left ventricular inner diameter (LVID)and the thickness of the left ventricular posterior wall(LVPW) in diastole and systole. From the aorta,recordings of the thickness of the medial wall(AoMW), lateral wall (AoLW) and the inner diameter(AoID) were obtained in diastole and systole.

Table 1. Outline of the project.

Group 1 2 3 4 5 6

CONTROL(n¼ 7)

EchocardiographyBW

BW BW EchocardiographyBW

BW EchocardiographyBWHistopathology

CATH(n¼ 6)

EchocardiographyBW

CatheterizationBW

RecoveryBW

EchocardiographyBW


SHAM(n¼ 7)

EchocardiographyBW

Sham surgeryBW

RecoveryBW

EchocardiographyBW


All mice were subjected to echocardiography in weeks 1, 4 and 6 of the experiment. In the second week, catheterized (CATH) and sham-operated (SHAM) mice were subjected to surgery, while control mice were left undisturbed. Body weights (BWs) were recorded weeklyfor all mice. At the end of the study, all mice were euthanized and the hearts were sampled for histopathology. CATH¼mice weresubjected to surgery with implantation of a carotid catheter. SHAM¼mice were subjected to surgery with ligation but not catheterizationof the carotid artery. CONTROL¼ control mice, not subjected to surgery.

Teilmann et al. 19

From these measurements, left ventricular volume indiastole (LV Vol-d) and systole (LV Vol-s), ejectionfraction (%EF) and fractional shortening (%FS) werecalculated using the software. Correct catheter tipplacement was confirmed for all catheterized miceusing the first post-surgical echocardiography (Figure2). All echocardiography recordings were completedwithin 20–30min between 09:00 and 12:00 h.

Pathology

After the final echocardiography, the mice were anesthe-tized with 5% isoflurane in 100% oxygen, and eutha-nized with isoflurane according to the AmericanVeterinary Medical Association (AVMA) guidelines.19

When death was confirmed, the hearts were sampledfor histopathology. The great vessels and associated tis-sues were removed before the hearts were weighed andimmersed in a buffered aqueous 4% formaldehyde solu-tion (Gurr� formaldehyde; VWR International, Vienna,Austria). The trimmed hearts were embedded in paraf-fin,20 and 4mm sections were cut on a rotary microtome(Thermo Scientific HM355S; Axlab, Vedbæk, Denmark)and stained with hematoxylin and eosin. The hearts wereserially transected on the transverse axis from base toapex with a 300mm distance for the evaluation of all thechambers. The sections were scored blinded by two inde-pendent pathologists, using a four-point scoring system,modified from Jokinen et al.21 (Table 2).

Each section was graded with respect to six param-eters: inflammation, perivascular inflammation, cardio-myocyte vacuolization, cardiomyocyte hyalinization,necrosis and fibrosis. Cardiomyocyte vacuolizationwas defined as a degenerative change with varyingdegrees of sarcoplasmic vacuolization. Cardiomyocytehyalinization was similarly regarded as a degenerativechange, where cardiomyocytes lost their patterns ofcross striations and attained a homogenous eosino-philic appearance. Necrosis of cardiomyocytes wasdefined as the presence of cells with homogenous sarco-plasm and pyknotic or karyorrhectic nuclei.Inflammation was characterized by the infiltration ofinflammatory cells within and/or between cardiomyo-cytes. The accumulation of perivascular inflammatorycells was scored as a separate parameter (perivascularinflammation). Fibrosis was defined as the presence ofcells with ovoid to elongated nuclei (fibroblasts) lyingwithin bands of eosinophilic fibrillar collagen, replacinglost cardiomyocytes.

Statistics

Data were analyzed using SPSS Statistics 19 (IBM,Armonk, NY, USA) and tested for normality, usingShapiro–Wilk’s test. Normally distributed datasetswere analyzed with a multivariate analysis of vari-ance (ANOVA) with ‘time’ and ‘group’ as fixedfactors, and with Tukey’s post hoc test. Where

Figure 1. Echocardiography. Examples are shown of M-mode ultrasound measurements of the heart in a cross-sectionalview (A) and aorta (C). Estimations of the thickness of the left ventricular anterior wall (LVAW), left ventricular posteriorwall (LWPW) and left ventricular inner diameter (LVID) are demonstrated in (B). From the aorta, estimations of thethickness of the medial wall (AoMW), lateral wall (AoLW) and the inner diameter (AoID) were equally obtained, asdemonstrated in (D).


appropriate, BW was included as a covariate(ANCOVA). Levene’s test for equality of varianceswas conducted to test that the error variances of the

dependent variables were equal across groups.Datasets that did not obey normality were subjectedto the Kruskal–Wallis test. Histology data were ana-lyzed using a multilevel generalized linear modelwith ‘Group’, ‘Mouse’ and ‘Observer’ as model fac-tors, where Mouse was defined as nested withingroups.22 This test examined the data for significanteffects both overall (omnibus) and for each modelfactor (Group, Observer and Mouse nested withinGroup) (Table 3).

To account for multiple comparisons, Bonferroni’scorrection was applied to the echocardiography andhistology data to avoid type 1 errors, resulting in anadjusted critical P value of 0.008, below which thesedata were considered to be significant. As for theweight data, P values below 0.05 were considered tobe significant.

Retrospective power calculations were performedusing echocardiography data from the control groupagainst the catheterized group, where the highest datavariation and the smallest difference between groupswere expected. With b¼ 0.20, a¼ 0.05 and standarddeviations (SDs) at 0.33 (cardiac measurements) and0.08 (aortic measurements), respectively, detectableeffect sizes were estimated to be 50% for cardiac meas-urements and 13% for aortic measurements, whichwere considered to be biologically relevant.

Results

Echocardiography

Please refer to Supplementary file 1 (supplementarymaterial can be found online at http://journals.sage-pub.com/home/lan) for the descriptive statistics. Themeasured variables (LVAW, LVID, LVPW in diastoleand systole, respectively) did not differ between groups(F(12, 92)¼ 1.243, P¼ 0.267) or over time (F(12,92)¼ 1.416, P¼ 0.173). Neither did the calculated vari-ables, LV Vol-d and LV Vol-s, differ between groups(F(12, 92)¼ 0.384, P¼ 0.966) or over time (F(12,92)¼ 1.357, P¼ 0.201).

Figure 2. Catheter tip placement in the proximal brachio-cephalic trunk. The top picture shows a B-mode echocardi-ography displaying the heart (H), ascending aorta (Ao) andbrachiocephalic trunk (Br) of one controlmouse. The bottompicture shows the same anatomic structures and correctcatheter placement (arrow) in one catheterized mouse.

Table 2. Four-point score system for histological grading of cardiac sections.

GradeSeverity oflesion Definition

1 Minimal A focus of less than 10 cardiomyocytes or with few countable inflammatory cells

2 Mild A focus larger than 10 cardiomyocytes but less than 250� 250 mm (estimated as half the width ofthe 40� objective)

3 Moderate A focus larger than 250� 250 mm but less than 500� 500 mm (estimated as the complete width ofthe 40� objective)

4 Severe An area larger than 500� 500 mm or inflammatory cells too numerous to count.

Hearts were sampled for histopathological evaluation and serial transverse sections were evaluated from each heart.

Teilmann et al. 21

The aortic measurements also did not differ betweengroups (F(12, 94)¼ 0.954, P¼ 0.498) or over time(F(12, 92)¼ 1.374, P¼ 0.192).

Weights

BWs did not differ between the groups (one-wayANOVA, F(2)¼ 0.371, P¼ 0.696). Likewise, therewere no significant group differences in the weights ofthe hearts (one-way ANCOVA, F(2)¼ 16.716,

P¼ 0.971) or the heart–BW ratios (Kruskal–Wallistest, �2(2)¼ 1.493, P¼ 0.474) (Table 4).

Histology

Comprehensive histological grading data are availablethrough Figshare.com (https://figshare.com/s/0e15c0c4716801045643; DOI: 10.6084/m9.figshare.4263569). Histological examples are illustrated with dif-ferent parameters in Figures 3 and 4.

Table 3. Statistics of histology data.

Model significance (P value)

Parameter Omnibus Group Mouse (Group) Observer

(3A)Cardiomyocyte inflammation <0.001 1.000 <0.001 0.058

Perivascular inflammation <0.001 0.215 <0.001 <0.001

Cardiomyocyte vacuolation <0.001 1.000 <0.001 <0.001

Cardiomyocyte hyalinization <0.001 0.548 <0.001 <0.001

Necrosis 0.122 1.000 1.000 0.998

Fibrosis <0.001 1.000 0.122 0.197

Parameter Observer Group-P Mouse (Group)-P Mean Median Min–Max

(3B)

Cardiomyocyte inflammation 1 1.000 0.475 0.11 0 0–4

2 1.000 0.022 0.15 0 0–2

Perivascular inflammation 1 0.815 <0.001 0.21 0 0–2

2 <0.001 <0.001 0.45 0 0–2

Cardiomyocyte vacuolation 1 0.011 <0.001 1.64 2 0–4

2 1.000 1.000 0.02 0 0–3

Cardiomyocyte hyalinization 1 <0.001 <0.001 1.31 1 0–4

2 1.000 <0.001 0.42 0 0–3

Necrosis 1 1.000 1.000 0.01 0 0–2

2 – – 0.00 0 0–0

Fibrosis 1 1.000 0.896 0.07 0 0–3

2 1.000 0.979 0.05 0 0–2

The top section (3A) presents the P values for each analyzed parameter, overall (omnibus) and for each model factor in a generalizedlinear model, where Group, Mouse and Observer were defined as model factors and where Mouse was defined as nested within groups.The bottom section (3B) presents P values, mean, median and minimum (Min) and maximum (Max) values for each of the parameters,separated by observer. To account for multiple comparisons, Bonferroni’s corrections were applied to avoid type 1 errors, resulting in anadjusted P value of 0.008. Significant P values are set in bold.

Table 4. Body weights (BWs), cardiac weights and heart–BW ratio.

CONTROL (n¼ 7) CATH (n¼ 6) SHAM (n¼ 7)

Body weight (g) 38.21� 2.30 39.60� 2.83 39.87� 5.45

Cardiac weight (g) 0.183� 0.017 0.179� 0.021 0.192� 0.028

Heart–BW ratio 0.478� 0.023 0.452� 0.031 0.489� 0.098

The table shows the mean (mean�SD) BW and cardiac weight (g) of control mice (CONTROL, n¼ 7), cathe-terized mice which had an arterial catheter implanted in their right common carotid artery (CATH, n¼ 6), andsham-operated mice which had their right common carotid artery ligated but not catheterized (SHAM, n¼ 7)as well as the heart–BW ratio. No significant group differences were found.


The omnibus test found overall significant effects inthe dataset for all the histological parameters, exceptfor cardiomyocyte necrosis (Table 3). These effects wereattributable to variation within groups of mice ratherthan between experimental groups. One parameter(fibrosis) was found to be significant in the omnibustest but not within or between groups of mice. Thiseffect was attributed to an interaction betweenGroups and Observers. No differences could be identi-fied between groups for any of the parameters.

For three of the parameters (perivascular inflamma-tion, cardiomyocyte vacuolation and cardiomyocytehyalinization) significant differences in the scoringswere found between the observers, with Observer 2 gen-erally scoring lower. When analyzing these parametersseparately for the two observers, an overall group dif-ference in perivascular inflammation was found byObserver 2, which was not significant for any of themodel factors. For cardiomyocyte hyalinization, a sig-nificant group difference could be identified byObserver 1, where the control mice had significantly

higher scores compared with the catheterized(P¼ 0.001) and sham-operated (P¼ 0.002) mice.

Discussion

Quick and easy vascular access is important in manymouse models, especially in drug discovery and safetytesting. Vascular catheters in mice, however, may beassociated with complications that threaten survival,complicate post-surgical catheter maintenance andimpair animal welfare.7,16,23 Thus, further efforts mustbe made to minimize deleterious effects on the models,and the redundant use of animals in research.

Carotid catheterization is a well-established proced-ure in mice.1,5,24 During surgery, the catheter isadvanced such that the tip is placed into a majorvessel in order to improve blood flow around the cath-eter and to minimize catheter patency failure.4 Thecatheter is advanced via the right common carotidartery down the brachiocephalic trunk, with the tippositioned in the proximal brachiocephalic trunk

Figure 3. Histological examples of inflammation. (A) Left ventricle. The perivascular spaces of two vessels are minimallyinfiltrated by few, countable mononuclear cells (perivascular inflammation, Grade 1). (B) Left ventricle. The lymphocyticinfiltrate surrounding this vessel is mild, consisting of more than a few countable cells but occupying less than250� 250 mm (Grade 2). (C–D) Base of heart, interventricular septum and aortic valves. The myocardium is severelyinfiltrated by lymphocytes and histiocytes, admixed with lesser numbers of neutrophilic granulocytes. The focus is largerthan 500� 500 mm (Grade 4). The area in the black box is shown in a higher magnification (40�) in (D). Bars¼ 100 mm in(A–B), bar¼ 200 mm in (C) and bar¼ 50 mm in (D).

Teilmann et al. 23

(as in the present study) or in the ascending or descend-ing aorta.3,25,26 Most published literature concerningthis technique, however, does not describe preciseplacement of the catheter tip, and thus some methodo-logical differences may exist between laboratories.

In the present investigation, a commercially avail-able standardized catheter was used, which has been

described previously.16 This catheter had a retentionbead one cm from the tip for securing the catheterinside the vessel during surgery. Based on our experi-ence, this also positioned the catheter tip in the prox-imal brachiocephalic artery, adjacent to the aortic archin NMRI mice of the applied age group (7–8 weeksold). Correct catheter placement was confirmed by

Figure 4. Histological examples of degenerative lesions. (A) Interventricular septum. A mild focus, occupying more thanthe space of 10 cardiomyocytes, but less than 250� 250 mm (Grade 2), consists of cells with ovoid to elongated nuclei(fibroblasts) lying within eosinophilic, fibrillar collagen (fibrosis). (B–C) Left ventricle. This area of fibrosis is moderate(larger than 250� 250 mm but less than 500� 500 mm, Grade 3). Fibroblasts lie within branching bands of fibrillar collagenand degenerative to necrotic cardiomyocytes (thick arrows). The area in the black box is shown in a higher magnification(40�) in (C). (D). Right ventricle. Focally cardiomyocytes appear to be hypereosinophilic with loss of the normal pattern ofcross striation (cardiomyocyte hyalinization, Grade 2). (E–F) Right ventricle. Focally, cardiomyocytes express varyingdegrees of sarcoplasmic vacuolation and necrotic changes, distinguished by the attainment of a homogenous appearance,sarcoplasmic fragmentation, pyknosis or karyorrhexis (thin arrows) (Grade 3). The area in the blue box (E) is shown in ahigher magnification (40�) in (F). Bars¼ 100 mm in (B, D and E) and bars¼ 50 mm in (A, C and F).


echocardiography (Figure 2). The aorta was avoideddue to the natural turbulence caused by blood ejectedfrom the left ventricle during systole, and in order tominimize direct effects on aortic arch baroreceptors. Insmall animals such as the mouse, however, a cathetertends to occupy a great part of even the larger vessellumens, and this may increase vascular resistance sys-temically, leading to increased cardiac pressure and adecrease in cardiac output.27

Pathological cardiac hypertrophy is a well-knownconsequence of altered hemodynamics, pressure orvolume overload,28 such as that caused by aortic con-striction.29,30 LV hypertrophy is one adaptation tochronic hemodynamic overloads on the heart,31 andmay lead to congestive heart failure and contractileimpairment.9,30 The present study was conducted toexamine potential pathological effects of carotid cath-eterization on cardiac physiology and function, due toan assumed effect on aortic hemodynamics from thepresence of the catheter. The sham-operated group,which had the carotid artery ligated but not catheter-ized, was included to account for the isolated effects ofcarotid ligation on hemodynamics and baroreceptoractivity.

Grossly, no differences in cardiac mass were foundbetween groups, either when comparing heart weightsor the heart–BW ratios, revealing no macroscopic evi-dence of cardiac hypertrophy for any of the mice.

Echocardiography recordings of the mouse heartswere used to assess potential physiological effectsfrom the long-term catheterization. In contrast to ourhypothesis, no group differences on the cardiac andaortic parameters could be identified using echocardi-ography. Anesthesia is known to relax vascular toneand baroreceptor reflexes, resulting in hypotension32,33

which may potentially affect the measurements.However, isoflurane anesthesia is generally known tohave minimal cardiodepressant effects in comparisonto other anesthetic agents,34,35 and as anesthetizingthe mice for the echocardiography procedures wasnecessary, a minor relaxation of vascular tone wasaccepted and was assumed to be equal across groups.Vascular tone was maintained steady by controlling theanesthetic depth during echocardiography.

On a cellular level, cardiomyopathy in mice normallyfollows a pattern of progression, beginning with cardi-omyocyte degeneration, which then advances to necro-sis, loss and replacement by fibrosis.21 In the presentstudy, lesions of degeneration and necrosis were oftenseen without concurrent fibrosis (Figures 4D–F), indi-cating that spontaneous alterations were more likely tobe a cause of normal background changes. However,fibrosis, indicating replacement of lost cardiomyo-cytes,36 was also noted on histology and was alwaysevaluated as potential cardiomyopathy with or without

concurrent degeneration or necrosis. The presence ofinflammatory infiltrates along with clear changes of car-diomyopathy is less common in mice,21 but wasincluded as a parameter in the present study due tothe implantation of a vascular catheter in these mice,which was regarded as a potential source of infection.

Contrary to our hypothesis, no effects of the experi-mental procedures could be identified with histology.Although the generalized linear analysis found overallsignificant effects in the model, these effects were attrib-utable to variation within the groups of mice and dis-crepancies between the two observers’ scorings for threeof the parameters. For only one of these parameters(cardiomyocyte hyalinization) a significant differencewas found between groups, where the control groupwas scored with a higher degree of cardiomyocyte hya-linization compared with the two experimental groups.Cardiomyocyte hyalinization was, in conjunction withcardiomyocyte vacuolation, included as a measure ofmyocardial degeneration and initial cardiomyopathy.Multifocal myofiber degeneration may, however,occur spontaneously.36 Thus, the higher scorings of car-diomyocyte hyalinization in control mice were attribu-ted to normal background variation. Although anobserver discrepancy was identified for three histo-logical parameters (perivascular inflammation, cardio-myocyte vacuolation and cardiomyocyte hyalinization),no significant group differences related to the experi-mental technique could be identified either collectivelyor in the observers’ respective score sets, and it is unli-kely that this discrepancy should have produced falsenegative results.

Based on the results in the present study, implant-ation of a carotid catheter with the tip positioned in theproximal brachiocephalic trunk has minimal influenceon cardiac and aortic physiology, and does not seem toinduce significant histopathological changes in theheart. These results are in contrast to histological ana-lyses obtained in a previous study,7 where early signs ofmyocardial degeneration with fibrosis were seen onlyfour days post-catheterization. In this study, catheter-ized mice were connected to an automated blood sam-pling device following surgery, and were subjected tocomputer-driven blood sampling and saline infusions.The present study also aimed to investigate the isolatedeffects of long-term carotid catheterization, and whythe catheter was capped following surgery and notaccessed until euthanasia. It is possible that the fre-quent access and utilization of the catheter in the pre-vious study increased cardiac strain, and furtherresearch may be warranted to fully examine the cardiaceffects of catheterization with regular access throughthe catheter.

In conclusion, long-term carotid catheterization withcatheter tip placement in the proximal brachiocephalic

Teilmann et al. 25

artery had no detectable effects on cardiac or aorticfunction in the present study.

Acknowledgements

The authors would like to thank Dr Cory Brayton and DrOtto Kalliokoski for thoroughly reading and commenting onthe manuscript.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of thisarticle.

Funding

The author(s) received no financial support for the research,

authorship, and/or publication of this article.

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Resume

La catheterisation de souris de laboratoire est couramment pratiquee en recherche biomedicale pour per-fuser des substances et des echantillons de sang. Une approche consiste a catheteriser l’artere carotidecommune droite et a avancer le catheter jusqu’a ce que l’embout soit place dans l’aorte ou le tronc brachio-cephalique proximal. Etant donne la petite taille du corps de la souris, un catheter occupe une grande partiedes lumieres de vaisseaux encore plus larges et peut augmenter la resistance vasculaire avec de potentielsimpacts pathophysiologiques sur le cœur. La presente etude comparait les fonctions cardiaques de souriscatheterisees, avec un placement de l’embout du catheter dans le tronc brachiocephalique, avec des sourispseudo-operees et des souris de controle non-operees. Des mesures echocardiographiques en mode M del’epaisseur de la paroi ventriculaire anterieure gauche, du diametre ventriculaire interne gauche et del’epaisseur de la paroi ventriculaire posterieure gauche ont ete effectuees pendant quatre semaines apresla catheterisation. Le volume ventriculaire gauche, la fraction d’ejection, le raccourcissement fractionnaireont ete calcules. De plus, les enregistrements aortiques de l’epaisseur des parois medianes et laterales ainsique du diametre interne ont ete mesures. En phase terminale, les cœurs ont fait l’objet d’une analysehistologique et leurs poids ont ete compares entre les groupes. Aucun effet sur les parametres de l’echo-cardiographie, l’histologie, les poids des corps ou les poids cardiaques n’a pu etre decele entre les groupes.Dans la presente etude, l’implantation d’un catheter dans la carotide avec le placement de l’embout ducatheter dans le tronc brachiocephalique proximal avait une influence minimale sur la physiologie cardiaqueet aortique et n’a pas entraıne de changements cardiaques significatifs.

Abstract

Katheterisierung von Labormausen findet in der biomedizinischen Forschung haufig Anwendung, umSubstanzen zu infundieren und Blut zu entnehmen. Ein Ansatz besteht in der Katheterisierung der rechtenKarotis und den Katheter so weit zu schieben, bis sich die Spitze in der Aorta oder im proximalen Truncusbrachiocephalicus befindet. Aufgrund der geringen Korpergroße der Maus nimmt ein Katheter einen großenTeil noch großerer Gefaßlumina ein und erhoht moglicherweise den Gefaßwiderstand mit potenziellen patho-physiologischen Folgen fur das Herz. Mit der vorliegenden Studie erfolgte ein Vergleich der Herzfunktionkatheterisierter Mause bei Positionierung der Katheterspitze im Truncus brachiocephalicus zwischen schei-noperierten Mausen und nicht-operierten Kontrollmausen. Vier Wochen lang wurden nach derKatheterisierung M-Mode-Echokardiographie-Messungen der Dicke der vorderen linksventrikularen Wand,des linksventrikularen Durchmessers und der Dicke der hinteren linksventrikularen Wand durchgefuhrt.Linksventrikulares Volumen, Auswurffraktion und Fractional Shortening wurden ermittelt. Außerdemwurden aortische Aufnahmen der Dicke der mittleren und seitlichen Wande sowie der Innendurchmessergemessen.

Teilmann et al. 27

Schließlich erfolgten eine histologische Analyse der Herzen sowie ein Vergleich der Korper- undHerzgewichte der Gruppen. Auswirkungen auf echokardiografische Parameter, Histologie, Korper- oderHerzgewicht wurden bei den Gruppen nicht festgestellt. In der vorliegenden Studie hatte die Implantierungeines Karotiskatheters mit Katheterspitzenpositionierung im proximalen Truncus brachiocephalicus minima-len Einfluss auf Herz- und Aorta-Physiologie und erzeugte keine signifikanten Herzveranderungen.

Resumen

La cateterizacion de ratones de laboratorio se realiza habitualmente en la investigacion biomedica paraaplicar sustancias y llevar a cabo muestras de sangre. Un metodo es cateterizar la habitual arteria carotidaderecha y hacer avanzar el cateter hasta que la punta este en la aorta o proxima al tronco braquiocefalico.Debido al reducido tamano corporal del raton, un cateter ocupa una gran parte de los lumenes del vasotodavıa mas grandes y puede aumentar la resistencia vascular con impactos patofisiologicos del corazon.Este estudio comparo la funcion cardıaca de ratones cateterizados, con la colocacion de la punta del cateteren el tronco braquiocefalico, con ratones con operacion simulada y con ratones de control sin operacion.Durante cuatro semanas despues de la cateterizacion, se realizaron mediciones ecocardiograficas en modo Mdel grosor de la pared anterior ventricular izquierda, del diametro interior ventricular izquierdo y del grosorde la pared posterior ventricular izquierda. Asimismo, se calculo el volumen ventricular izquierdo, la fraccionde expulsion y el recorte fraccional. Tambien se realizaron mediciones de los registros aorticos del grosor delas paredes laterales y medias ademas del diametro interior. Finalmente, se realizaron analisis histologicosde los corazones y se compararon los pesos corporales y los pesos del corazon entre los grupos. No seencontraron efectos en los parametros ecocardiograficos, histologicos, de los pesos corporales o los pesoscardıacos entre los grupos. En este estudio, la implantacion de un cateter carotıdeo con la colocacion de supunta proxima al tronco braquiocefalico tuvo una influencia mınima en la fisiologıa cardıaca y aortica y noindujo cambios cardıacos significativos.


ab oratory

l i m i t e d

lan imals

Original Article

Diurnal rhythms of blood glucose, serumghrelin, faecal IgA and faecalcorticosterone in rats subjected torestricted feeding using the diet board

Iiris Kasanen1, Katja Inhila1, Eriika Savontaus2,Hanna-Marja Voipio3, Kai Okva1, Satu Mering1, Vesa Kiviniemi4,Jann Hau5, Sakari Laaksonen3 and Timo Nevalainen1

AbstractLaboratory rats are generally fed ad libitum, although this method is associated with obesity and an increasedfrequency of spontaneous tumours. It has been challenging looking for ways to limit feed consumption ingroup-housed rats without any setbacks to animal welfare and scientific results. The diet board, as a methodof dietary restriction, was used in the present study. Diet board feeding allows group housing and shouldresult in enhanced welfare compared with traditional methods of dietary restriction. With respect to animalmodel robustness and translatability of results it is important that the feeding regime does not affect diurnalrhythmicity of biological parameters. In the present study the effects of diet board feeding on diurnal rhythmsof blood glucose, serum ghrelin, faecal immunoglobulin A (IgA) and faecal corticosterone were assessed. Thediet board did not alter diurnal rhythms, and adds weight to the use of this method for dietary restrictionwhich should benefit animal health and the validity of scientific results generated from the animals.

Keywordsrats, restricted feeding, diurnal rhythms, refinement, reduction

Date received: 31 January 2017; accepted: 23 April 2017

The world is not yet perfect, and certainly not when itcomes to feeding laboratory rodents. The negativeaspects of ad libitum feeding include a high variationin food intake,1 and consequently a high variation inbody weight and other variables. It can also result inhigh mortality and morbidity.2–5 Dietary restrictiondoes solve these problems,6–8 but it also creates newones. The most common method of implementing diet-ary restriction in rodents is to house them singly andgive them a pre-calculated meal once a day, oftenduring the daytime.9 However, this practice compro-mises both animal welfare and scientific integrity. Thelack of conspecifics is a serious threat to the wellbeingof rats.10,11 Feeding during the daytime disturbs thediurnal rhythms of physiology and behaviour, thusconfounding research results.12–14 When comparingresults obtained from animals fed ad libitum withthose subjected to dietary restriction, it is not clearwhether the possible differences reflect decreased caloricintake per se, or altered diurnal rhythms.

We have developed a novel method of dietaryrestriction. The diet board15,16 offers the possibility ofcombining dietary restriction with group housing andnormal eating rhythms. The diet board is made up oftwo wooden boards onto which food pellets areembedded.15,16 The diet board is always in the cage,allowing the rats to eat as often as they choose.

1Laboratory Animal Centre, University of Eastern Finland, Kuopio,Finland2Department of Pharmacology, Drug Development andTherapeutics and Turku Center for Disease Modeling, Universityof Turku, Turku, Finland3Laboratory Animal Centre, University of Oulu, Oulu, Finland4IT Centre, University of Eastern Finland, Kuopio, Finland5Department of Experimental Medicine, University of Copenhagenand University Hospital of Copenhagen, Copenhagen, Denmark

Corresponding author:T Nevalainen, Laboratory Animal Centre, University of EasternFinland, Kuopio Campus, PO Box 1627, 70211 Kuopio, Finland.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 29–37



sagepub.co.uk/


DOI: 10.1177/0023677217709850


However, the rats need to gnaw wood before reachingthe pellets. Their food intake is decreased by 15% com-pared with ad libitum fed controls. We have previouslyshown that diet board feeding causes a 15% decrease inweight gain, a 30% decrease in gonadal fat mass, a 3%decrease in skeletal growth, and elicits similar endocri-nological changes as in traditional methods of dietaryrestriction.15–18

The aim of the present study was to investigate howdiet board feeding affects diurnal rhythms of physiolo-gical variables. In order for the diet board to be gen-erally accepted as a refinement alternative to thetraditional methods of dietary restriction, it shouldmeet two conditions: (i) Diet board feeding shouldresult in enhanced health and welfare compared withtraditional methods of dietary restriction. (ii) The qual-ity of results obtained with the diet board should be asgood as or better than in experiments using other meth-ods of dietary restriction.19 To ensure scientific qualityand relevance, undisturbed diurnal rhythms would be amajor achievement in the practice of restricting caloricintake in rodents. Our hypothesis was that dietboard feeding does not alter diurnal rhythms of phy-siological variables in animals compared with ad libi-tum feeding.

Animals

The study was performed at the Laboratory AnimalCentre, University of Eastern Finland, Finland. Thestudy plan was reviewed and approved by the FinnishNational Ethics Committee (licence number ESLH-2008-03945/Ym23).

A total of 48 barrier bred HsdBrlHan:Wist male rats(Laboratory Animal Centre, University of EasternFinland, Kuopio, Finland) were used in this study.The barrier was free of the pathogens listed in theFederation of European Laboratory Animal ScienceAssociations (FELASA) recommendations for healthmonitoring.20 The animals were 12 weeks old at thecommencement of this 10-week study. The rats wereidentified with ear notching.

Animal housing

All animals were kept in one animal room, housed ingroups of three in solid bottom Makrolon� polycarbo-nate cages (55� 35� 20 cm; Tecniplast, Buguggiate,Italy) with a wire-grid hopper (Bayer AG,Leverkusen, Germany). The cage floor was coveredwith 300 g of aspen chip bedding (Tapvei Ltd, Kaavi,Finland). Tap water in polycarbonate bottles was avail-able ad libitum. Each cage was provided with threeaspen chewing blocks (Tapvei Ltd). The cages andchewing blocks were changed weekly, and the bottles

twice a week. The room temperature was 21� 1�C andrelative humidity 55� 15%. The lights came on at07:00 h and off at 19:00 h. The light intensity in thecages was approximately 130 lx. All animals wereweighed twice a week (Sartorius 1B31; Sartorius-Werke GmbH, Gottingen, Germany).

Feeding

All animals were fed autoclaved (121�C, 20min,220 kPa, Finn-Aqua 121821 D; Steris Finn-Aqua,Tuusula, Finland) Lactamin R36 pellets(Lantmannen, Kimstad, Sweden). The control animals(n¼ 24) were fed ad libitum from the food hopper. Thestudy group (n¼ 24) was fed solely from the dietboard.15 The diet board consisted of two crossedaspen boards (36.0� 12.2� 2.7 cm) with 20 verticallydrilled holes (Ø 12.5mm) filled with food pellets andfixed on site by autoclaving. Each hole had a 2mm wideslot showing the pellets, thereby providing rats with aneasier access to the food. Two corners of each board(6.0� 6.0 cm) were removed. Control animals had asimilar board of the same shape and size, but withoutthe holes or food. Both boards were changed to newones at the cage change stage.

Study design

The animals were randomly allocated into 16 cageswith three rats in each. The cages were randomlydivided into eight control cages (ad libitum feeding)and eight study cages (diet board feeding). The cageswere placed on tables in the animal room, all at thesame height. The study was arranged in two equalblocks with a two-week phase shift.

Sampling

Blood samples were collected six times at 4 h intervalsbeginning at 09:00 h on week 9. The samples were takenby piercing a hole in the vena saphena with a 25Gneedle21 and the resulting blood drops were collectedinto a test tube. The sample volumes did not exceedthose recommended by Diehl et al.22

Humane endpoints

Three different humane endpoints were established forthe different phases of the experiment. During the firstweek of the experiment the limit was set to 15% weightloss. During experimental weeks 2 to 4, a failure to gainweight over a two-week period was added to theendpoint. From the fifth week onwards, weight loss of5% or more during any one week was the endpoint.Dehydration, abnormal behaviour or appearance,


or unexpected trauma or diseases were considered asgeneral criteria for exclusion from the study at all times.


Sample assays

Blood glucose concentrations were measured instantlyafter drawing the blood with Accu-Chek Compact Plus(Roche Diagnostics, Mannheim, Germany). The bloodintended for serum ghrelin analysis was allowed to coa-gulate for 10 to 15min, and was then centrifuged at12 000 rpm for 15min at room temperature (Eppendorf5412; Eppendorf, Hamburg, Germany). The serumattained was placed into plastic tubes and immediatelyfrozen at –20�C, and then shipped frozen to an assaylaboratory where the samples were kept at –80�C untilanalysis. The concentrations of serum ghrelin were mea-sured using a commercial kit (total ghrelin RIA kit;Linco Research, St Charles, MO, USA).

Faecal samples were collected four times a day fromeach cage at 6 h intervals beginning at 14:00 h on week6. On collection day, the cage floor was covered withonly half the amount of normal bedding to make thesample collection easier. At each time point all faecalpellets among the bedding were carefully collected withforceps and stored at –20�C. The extraction of immu-noglobulin A (IgA) and corticosterone from the faecalsamples was carried out according to the methoddescribed by Pihl and Hau.23 The concentrations ofIgA (rat IgA quantitation kit; Bethyl Laboratories,Montgomery, TX, USA) and corticosterone (DRGDiagnostics, Marburg, Germany) were analysed withcommercial kits using a Multiskan EX microtiterreader (Thermo Electron Corp, Waltham, MA, USA).

Statistics

The appropriate size of the experiment was estimatedwith the resource equation method,24 and the degree offreedom for error was 13. The differences between thediet board and ad libitum groups were analysed withlinear mixed models for repeated measures using allcollected data. Time, treatment and their interactionwere included as fixed effects into the model. Arandom cage effect was included in the models inorder to accommodate for the possibility of correlatedoutcomes among cagemates. The linear mixed model’sassumption of normality of distribution was tested withShapiro–Wilkinson’s test from the residual varianceterms. Where necessary, the variables were log-trans-formed to normalize their distributions.

The results of normally distributed variables (corti-costerone, glucose) are presented as model-based esti-mates of the arithmetic means and 95% confidence

intervals (CI). For variables that were not normallydistributed (IgA, ghrelin), the results are presented asmodel-based estimates of the geometric means and their95% CI. In the graphic representation of the normallydistributes, variable means and standard deviationsfrom the raw data are used. For the variables not nor-mally distributed the means and 95% CI from the rawdata are used. Due to a technicality, the values in thegraphic representation of the body weight constitute anexception: there the values are model-based estimates ofthe means and their 95% CI, even though the weightswere normally distributed.

The statistical software package used to process andanalyse the data was SPSS 14.0 for Windows (SPSSInc, Chicago, IL, USA). The graphs were drawn withSigmaPlot 10.0 (Systat Software Inc, San Jose, CA,USA).

Results

Diet board fed animals resulted in significant differ-ences (P¼ 0.016) in their body weights compared withad libitum fed animals, with the diet board fed animalsshowing an approximately 10% lower body weightthroughout the experiment (Figure 1). None of the ani-mals reached the humane endpoints set for thisexperiment.

With ghrelin, glucose, IgA and corticosterone, timewas a significant factor (P< 0.05), i.e. the variablesshowed a diurnal rhythm. There were no statisticallysignificant differences between the ad libitum and thediet board groups in any of the measured variables(P> 0.05). The interactions between time and treat-ment were not significant in any of the variables(P> 0.05), indicating that there were no significant dif-ferences in the diurnal rhythms of ad libitum and dietboard fed rats (Figures 2–5). The model-based esti-mates of the means and their 95% CI at each timepoint are presented in Table 1.

Discussion

The diet board was developed as a refinement alterna-tive to traditional methods of dietary restriction. Novelmethods should always be validated before they can berecommended for wider use or offered for commercialproduction. Methods intended for laboratory animaluse should also be evaluated with respect to theirimpact on the 3Rs (replacement, reduction and refine-ment).19 The effects on the quality and relevance of theresearch data obtained from rats subject to diet boardfeeding should also be established. We have previouslyshown the diet board’s efficacy, safety and reproduci-bility in providing moderate caloric restriction forlaboratory rats.15–17 The most important refinement

Kasanen et al. 31

Figure 1. Body weights of diet board and ad libitum fed rats throughout the 10-week experiment. The error barsrepresent the 95% confidence intervals of the model-based estimates of the means. The figure is based on datafrom 48 rats.

Figure 2. Diurnal rhythms of blood glucose concentration in diet board and ad libitum fed rats on study week 9. The errorbars represent standard deviations from the raw data. The horizontal black bar represents the dark period during the 24 hsampling. The figure is based on data from 48 rats.


Figure 4. Diurnal rhythms of faecal corticosterone secretion (amount of corticosterone secreted per cage per hour) indiet board and ad libitum fed rats on study week 6. The error bars represent standard deviations from the raw data. Thehorizontal black bar represents the dark period during the 24 h sampling. The figure is based on data from 16 rat cages.

Figure 3. Diurnal rhythms of serum ghrelin concentration in diet board and ad libitum fed rats on study week 9. The errorbars represent the 95% confidence intervals of the means of the raw data. The horizontal black bar represents the darkperiod during the 24 h sampling. The figure is based on data from 48 rats.

Kasanen et al. 33

Table 1. Values of blood glucose, serum ghrelin, faecal IgA and faecal corticosterone.

Variable

Ad libitum Diet board Overall P value

Mean95% CILower

95% CIUpper Mean

95% CILower

95% CIUpper Treatment Time

Time *treatment

Blood glucose (mmol/L)09:00 h 5.2 5.1 5.5 5.2 5.0 5.4 0.7 <0.001 0.6

13:00 h 5.7 5.5 5.9 5.6 5.3 5.8

17:00 h 6.1 5.9 6.3 6.0 5.7 6.2

21:00 h 6.3 6.1 6.5 6.3 6.1 6.5

01:00 h 6.4 6.2 6.6 6.4 6.2 6.6

0:500 h 6.2 6.0 6.4 6.4 6.2 6.6

Serum ghrelin (pg/mL)09:00 h 1440 1300 1600 1420 1280 1580 0.4 <0.001 0.2

13:00 h 1420 1270 1570 1290 1150 1430

17:00 h 1310 1180 1450 1260 1140 1400

21:00 h 1220 1100 1360 1230 1110 1360

01:00 h 1420 1280 1580 1260 1140 1400

05:00 h 1480 1330 1640 1420 1280 1570

Faecal CORT (ng/h/cage)08:00–14:00 h 230 160 300 240 170 320 0.6 0.02 0.3

14:00–20:00 h 190 110 260 210 130 280

20:00–02:00 h 270 190 340 350 280 420

02:00–08:00 h 310 240 390 260 180 330

(continued)

Figure 5. Diurnal rhythms of faecal immunoglobulin A (IgA) secretion (amount of IgA secreted per cage per hour) in dietboard and ad libitum fed rats on study week 6. The error bars represent the 95% confidence intervals of the means of theraw data. The horizontal black bar represents the dark period during the 24 h sampling. The figure is based on data from16 rat cages.


the diet board can offer compared with traditionalmethods of dietary restriction is the possibility ofgroup-housing the animals. Preference tests indicatethat the company of conspecifics is valued more byrats than any other form of environmental enrich-ment,25 and both European and American guidelinesclearly state that rodents should be group-housed bydefault, and single housing should only be practisedfor veterinary, welfare or scientific reasons for a limitedtime period.26,27 Furthermore, the diet board also pro-vides a recommended structural complexity into thecage.27

We have previously shown that the diet board elicitssimilar, but somewhat milder, endocrinological effectsas using traditional methods of dietary restriction.16

This further proves that the diet board provides truecaloric restriction, and not merely as an enrichment forthe animals.

The purpose of this study was to assess the effects ofdiet board feeding on diurnal rhythms of blood glucose,serum ghrelin, faecal IgA and faecal corticosterone.These are variables known to have a clear diurnalrhythm that can be altered by changes in feeding sche-dules.28–34 Disruption of diurnal rhythms is one of themost important threats to research quality when usingtraditional methods of dietary restriction. Our hypoth-esis was that since the animals are free to eat when theychoose, the diet board would not alter diurnal rhythms.The results of this study proved our hypothesis to becorrect. Diet board feeding did not cause significantchanges in diurnal rhythms or the actual levels of theanalysed variables (Figures 2–5). The diet board is thusa refinement alternative in the best sense of the word,providing both enhanced welfare and better science.

The reduction potential of using the diet board hasalso been evaluated. We have investigated the equalityof variances from residual variance terms in more than20 different variables from different experiments with

the diet board. No significant differences were found inthe variances of any of the variables. Compared with adlibitum feeding, diet board feeding does not alter thenumber of animals needed in experiments in order tomaintain the same statistical power.

In conclusion, the diet board is now sufficiently vali-dated as a method of dietary restriction, as well asproviding a structural complexity to the cage environ-ment. It can therefore be recommended for group-housed rats when dietary restriction is needed.


The author(s) declared no potential conflicts of interest with

respect to the research, authorship, and/or publication of thisarticle.

Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship, and/or publication of thisarticle: The authors received funding from the Academy of

Finland and the Ministry of Education, Finland.

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Table 1. Continued

Variable

Ad libitum Diet board Overall P value

Mean95% CILower

95% CIUpper Mean

95% CILower

95% CIUpper Treatment Time

Time *treatment

Faecal IgA (ng/h/cage)08:00–14:00 h 27800 20700 36800 27600 20700 36800 0.5 <0.001 0.4

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02:00–08:00 h 45800 34300 61000 33500 25100 44700

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Resume

Les rats de laboratoire sont generalement nourris ad libitum bien que cette methode soit associee a l’obesiteet a une frequence accrue de tumeurs spontanees. La recherche de moyens de limitation de la consommation


d’aliments chez les rats vivants en groupe sans effets negatifs sur le bien-etre des animaux et les resultatsscientifiques a ete difficile. Le tableau de regime en tant que methode de restriction alimentaire a ete utilisedans la presente etude. L’alimentation dans le cadre d’un regime alimentaire est favorable a l’habitat collectifet devrait entraıner l’amelioration du bien-etre par rapport aux methodes traditionnelles de restriction ali-mentaire. En ce qui concerne la robustesse du modele animal et la traduction des resultats, il est importantque le regime d’alimentation n’affecte pas la rythmicite diurne des parametres biologiques. Dans la presenteetude, les effets de l’alimentation dans le cadre d’un regime alimentaire en fonction des rythmes diurnes dela glycemie, de la ghreline, de l’IgA fecale et de la corticosterone fecale ont ete evalues. Le tableau de regimen’a pas modifie les rythmes diurnes et a ajoute du poids a l’utilisation de cette methode pour une restrictionalimentaire au benefice de la sante animale et la validite des resultats scientifiques generes par les animaux.

Abstract

Laborratten werden gemeinhin ad libitum gefuttert, allerdings ist diese Methode mit Fettleibigkeit underhohter Haufigkeit von Spontantumoren verbunden. Die Suche nach Moglichkeiten zur Einschrankung desFutterverbrauchs bei Ratten in Gruppenhaltung ohne Beeintrachtigung von Tierwohl und wissenschaftlichenErgebnissen hat sich bisher als schwierig erwiesen. Diet Board- Futterung ermoglicht Gruppenhaltung undsollte gegenuber herkommlichen Methoden der Futterrationierung in optimiertem Tierwohl resultieren. InBezug auf Tiermodell-Robustheit und Ubertragbarkeit von Erkenntnissen gilt es zu vermeiden, dass dasFutterungskonzept den Biorhythmus biologischer Parameter beeinflusst. In der vorliegenden Studiewurden die Auswirkungen der Diet Board-Futterung auf den Biorhythmus von Blutglukose, Serumghrelin,fakalem IgA und fakalem Corticosteron untersucht. Das Diet Board fuhrte zu keinen Veranderungen derBiorhythmen, weshalb dem Einsatz dieser Methode zur Futterbeschrankung im Interesse des Tierwohlsund der Gultigkeit der durch die Tiere gewonnenen wissenschaftlichen Erkenntnisse durch diese StudieGewicht verliehen wird.

Resumen

Las ratas de laboratorio por lo general son alimentadas ad libitum aunque este metodo esta asociado a laobesidad y a un aumento frecuente de tumores espontaneos. Es complicado buscar formas para limitar elconsumo de alimentos en ratas enjauladas en grupo sin complicar el bienestar animal y los resultadoscientıficos. En el presente estudio se utilizo una tabla de dieta como metodo de restriccion alimenticia. Laalimentacion con tabla de dieta permite el enjaulamiento en grupo y deberıa mejorar el bienestar animal encomparacion con otros metodos tradicionales. En cuanto a la solidez del modelo animal y la traducibilidad delos resultados, es importante que es regimen de alimentacion no afecte el ritmo diurno de los parametrosbiologicos. En el presente estudio, se evaluaron los efectos de la alimentacion con tabla de dieta sobre losritmos diurnos de la glucosa en sangre, grelina en suero, IgA fecal y corticosterona fecal. La tabla de dieta noaltero los ritmos diurnos y dio mas peso al uso de este metodo para la restriccion dietaria para el beneficio dela salud animal y la validez de los resultados cientıficos generados por los animales.

Kasanen et al. 37

ab oratory

l i m i t e d

lan imals

Original Article

Diagnostic imaging modalitiesand surgical anatomy of thetemporomandibular joint in rabbits

Michal Kyllar1,2, Barbora Putnova3,4, Vladimır Jekl5,Ladislav Stehlık6, Marcela Buchtova3 and Jan Stembırek3,7

AbstractThe temporomandibular joint (TMJ) is a condylar synovial joint that, together with the masticatory muscles,controls mandibular movement during mastication. The rabbit is often used as a model species for studyingthe mechanisms of TMJ diseases, and in regenerative research. However, there are significant differencesbetween rabbit and human TMJs that should be taken into account before using this model for experimentalresearch. Here, we use several analytical approaches (radiography, computed tomography and magneticresonance imaging) to enable a detailed description and analysis of the rabbit TMJ morphology. Moreover,possible surgical approaches have been introduced with a focus on available access into the rabbit TMJ cavity,which relate our findings to clinical usage.

Keywordsanimal model, feeding, jaw, rabbit, temporomandibular joint (TMJ)

Date received: 24 September 2016; accepted: 3 March 2017

The temporomandibular joint (TMJ, articulatio tem-poromandibularis) is a multi-component structure that,together with the masticatory muscles, controls man-dibular movement during mastication. It consists ofthe condylar process of the mandibular ramus (ramusmandibulae), mandibular fossa of the temporal bone(fossa mandibularis ossis temporalis), a thin articulardisc (discus articularis), and a loose joint capsule rein-forced with fibrous lateral ligaments.1 The structureand function of this incongruent, condylar synovialjoint (hinge sliding joint) are unique among the dia-rthrodial joints.2

There are many diseases and traumas affecting theTMJ. While many studies have provided detailedinformation about the development, anatomy andaetiopathogenesis of TMJ diseases, there is a lack ofcomprehensive understanding of the mechanism initiat-ing these diseases. Many of the symptoms are difficultor impossible to treat using current methods, highlight-ing the need for extensive research in this field. The useof animal models for such research is indispensable.3

Due to physiological and anatomical differencesbetween the human TMJ and those of experimentalanimals, no one animal model can provide a full under-standing that could be directly related to humans.4 In

addition, many other variables, such as gender, age, sizeand depth/age of the defect, postoperative treatment,etc., need to be considered when designing experiments.Therefore, an investigator faces the challenging task ofchoosing the most suitable model and treatment proto-col in order to answer a specific question. Discrepancies

1Department of Anatomy, Histology and Embryology, Faculty ofVeterinary Medicine, University of Veterinary and PharmaceuticalSciences Brno, Brno, Czech Republic2Companion Care, Broadstairs, UK3Institute of Animal Physiology and Genetics, v.v.i., Academy ofSciences of Czech Republic, Brno, Czech Republic4Department of Pathological Morphology and Parasitology,University of Veterinary and Pharmaceutical Sciences Brno,Brno, Czech Republic5Avian and Exotic Animal Clinic, University of Veterinary andPharmaceutical Sciences Brno, Brno, Czech Republic6Department of Diagnostic Imaging, Small Animals Clinics, Facultyof Veterinary Medicine, University of Veterinary andPharmaceutical Sciences Brno, Brno, Czech Republic7Department of Oral and Maxillofacial Surgery, University HospitalOstrava, Czech Republic

Corresponding author:Marcela Buchtova, Institute of Animal Physiology and GeneticsCAS, v.v.i., Veveri 97, Brno, 602 00, Czech Republic.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 38–50



sagepub.co.uk/


DOI: 10.1177/0023677217702178


in the results of clinical studies focused on treatmentmodalities for TMJ dysfunction make it obvious thatanimal models are needed to deEne and control experi-mental variables.5 Although no one animal model canexactly duplicate the human condition, a number ofauthors have argued that, despite its prognathism, thepig, Sus scrofa, is the best non-primate model for mod-elling the human TMJ form and function.6–9Althoughlarge animal models such as the pig may resemblehumans more closely than smaller animals, they areusually neither practical nor economically feasible forconducting initial experiments in large animals.10

One of the smaller animal models frequently utilizedin research is the rabbit. Rabbits are easy to handle andinexpensive to maintain in cages, making them conveni-ent animals for use in experimental studies. Rabbits areoften used in studies to test dental implants,11–14 to testbone grafts following the extraction of cheek teeth,15

and to investigate the influence of biomaterials on thebone healing process around the tooth sockets.16,17

Studies on the effect of tooth loss on the histochemicalcomposition of TMJ cartilage and disc18 and on thehistology of the condyle19 have been performed.Surgical alterations of the rabbit TMJ for investigationof discectomy effects20,21 and implantation of discreplacements have also been reported.22 However,although the rabbit has been routinely used as anexperimental animal model, very little information isavailable about the anatomy of its TMJ, and its cover-age in veterinary textbooks is even more cursory.23

The best description of the rabbit TMJ24 is unfortu-nately not easily accessible to English-speakingresearchers; and other descriptions of rabbit TMJs arecontradictory, particularly with respect to theirnomenclature.25

Considering the lack of complete information aboutthe anatomical and histopathological characteristics ofthe rabbit TMJ, the purpose of this investigation was to(1) provide an overall description of a rabbit TMJ withan emphasis on gross anatomy and its comparison to ahuman TMJ; (2) describe the appearance of theanatomical structures on radiographs, computed tom-ography (CT) and magnetic resonance imaging (MRI)scans, and discuss the advantages and disadvantages ofindividual techniques for TMJ studies; and (3) relateour Endings to clinical usage and describe achievablesurgical approaches into a rabbit TMJ.


Animals

Twelve clinically normal eight-month-old outbred maleNew Zealand white specific pathogen-free rabbits(Oryctolagus cuniculus, strain Hsdlf:NZW; Harlan

Laboratories Inc, Belton, UK) were used in thisstudy. The weight of the animals ranged from 2.92 to3.15 kg (mean 3.03� 0.2 kg). The animals were fullydeveloped and were free of any pathological processes.All the rabbits were housed individually in an animalcare facility under controlled conditions, and werehandled and euthanized according to the agreementof the Branch Commission for Animal Welfare of theMinistry of Agriculture of the Czech Republic (projectPP52-2013 UVPS).

Diagnostic imaging

The rabbit heads were radiographed using a GierthX-ray machine (Gierth HF 200A; Gierth X-RayInternational GmbH, Riesa, Germany). Radiographswere captured on computed radiography cassettes.The focal length was set to 100 cm and the exposurevalues were 50 kV, 200mA, and 150ms (30mAs). Theimages were stored in a digital imaging and communi-cations in medicine (DICOM) format using a computedradiography system (FCR Capsula XL; Fuji, Tokyo,Japan). The image resolution was 1760� 2140 pixels.

Native transverse CT scans of each rabbit’s headwere obtained using a multidetector CT scanner(LightSpeed 16; GE Medical Systems, Milwaukee,WI, USA). The scanning protocol was 80 kV, with anautomatic mA setting, a tube rotation time of onesecond, a slice thickness of 1.25mm, a spiral pitchfactor of 0.9 and a high frequency convolution kernel(proprietary name: bone). All the scans were obtainedusing the helical mode. Radiographic and CT analyseswere performed at the Department of DiagnosticImaging, Small Animals Clinic, Faculty of VeterinaryMedicine (Brno, Czech Republic).

MRI analysis (Bruker Avance 9.4T, Bruker BioSpec;Bruker, Ettlingen, Germany) of the rabbit heads wasconducted at the Institute of Scientific Instruments ofthe Czech Academy of Sciences (Brno, CzechRepublic). Images were acquired in sagittal and trans-verse planes with fast spin echo (FSE) sequences. Theheads were positioned in ventral recumbency during thescanning procedure. FSE T1-weighted transverse MRimages were obtained with the following parameters:FLASH, AVG¼ 6, TE¼ 3.7ms, TR¼ 369ms, matri-ces¼ 512� 512. Acquisition time 14.5min and RARE,AVG¼ 6, TE¼ 25ms, TR¼ 3500ms, matrices¼512� 256, RARE factor¼ 8.

Dissection procedure

Palpable anatomical landmarks were identified prior todissection using a rabbit skull model as a reference.Anatomical dissections of the TMJ were performedusing a stratigraphic approach, and each dissected

Kyllar et al. 39

layer was photographed using a Nikon D 5100 camera(Nikon Europe BV, Amsterdam, The Netherlands).The key anatomical structures were identified and indi-cated on the photographs.

Histological evaluation

The harvested mandibular condyles (condyles mandibu-lares), including the articular cartilage, were fixed forthree days in 10% buffered formaldehyde and decalci-fied in 12.5% EDTA solution (pH¼ 8), which was pre-pared as a mixture of 0.5mmol/L EDTA, 4% bufferedformaldehyde and PBS buffer (pH¼ 7,4). The tissuesamples were left in this decalcification solution in anincubator at a temperature of 37�C for 11 months.Following the decalcification, the samples were pro-cessed for histological analysis using a standard proced-ure – they were washed in water (overnight) anddehydrated in an increasing alcohol series (30% for3 h; 50% for 3 h; 70% overnight; 80% for 2 h; 95%for 2 h; 100% for 1 h). Then, the samples were soakedin xylene for 5 h, run through three paraffin baths(for 1 h in each of them) and embedded in paraffinblocks.

Five micrometre paraffin sections were stained withhaematoxylin and eosin (H&E), Alcian blue (AB) andMasson’s trichrome.26 The staining protocols used inthis study were modified in the following steps: after

staining in Mayer’s haematoxylin, staining in acid–alcohol was not differentiated but was left in warmwater for 5min. In the case of the AB staining, H&Ewas used for a counterstaining. In the case of theMasson’s trichrome, Weigert’s haematoxylin (DiapathSpA, Martinengo BG, Italy) was used, then the sectionswere differentiated in acid–alcohol, stained withFuchsin Ponceau acc. Masson (Diapath SpA), differen-tiated in 1% phosphotungstic acid and stained with alight green (Diapath SpA).

Morphometric assessment

Morphometric assessment was carried out on the dis-sected and macerated skulls (Figure 1). TMJ width(TMJW) was determined by measuring the distancebetween the medial aspect of the zygomatic process ofthe temporal bone (processus zygomaticus ossis tempor-alis) and the squama of the temporal bone. TMJ length(TMJL) was measured as the distance between the ros-tral aspect of the zygomatic process of the temporalbone and the most caudal projection of the zygomaticarch (arcus zygomaticus). Condylar width (CW) wasmeasured at its widest point.

Furthermore, a surface projection of the TMJ on therabbit skull was established by using an axis of thezygomatic arch and the line connecting the caudalaspect of the zygomatic process of the frontal bone

Table 1. Origins and insertions of muscles connected with temporomandibular joint (TMJ) function.

Muscle Subdivision Origin Insertion

Musculus masseter Pars superficialis Arcus zygomaticus – rostralportion

Angulus mandibulae

Pars profunda rostralis Arcus zygomaticus – mediallyon caudal portion

Caudal corpus mandibulae tothe level of angulusmandibulae

Pars profunda caudalis Arcus zygomaticus – mediallyon caudal portion

Angulus mandibulae to distalpart of ramus mandibulae

Musculus temporalissuperficialis

Os temporale and os parietale Lateral and proximal aspectof the processus coronoi-deus mandibulae

Musculus temporalisprofundus

Pars medialis Os temporale within the orbit Rostral ridge of the coronoidprocess

Pars lateralis Os temporale Proximal ridge and medialaspect of the coronoidprocess

Musculus pterygoideusmedialis

Os pterygoideum/fossapterygoidea

Medial aspect of the ramusmandibulae distally, angu-lus mandibulae and corpusmandibulae caudally

Musculus digastricus Processus jugularis ofthe occipital bone

Rostral extent of the angulusmandibulae


(processus zygomaticus ossis frontalis) and ear canal(meatus acusticus externus).

Histological samples of the head of the mandible(caput mandibulae) were stained using AB and wereused for measurements of the cartilage thickness.Photodocumentation was taken using a DMC2900camera (Leica, Wetzlar, Germany), and the thicknessof the hyaline cartilage was measured using theAxioVision 40 version 4.8.2.0 software (Carl ZeissMicroImaging GmbH, Munich, Germany). The

acquired data were processed using the Statistica soft-ware (StatSoft, Tulsa, OK, USA).

Results

The TMJ is a complex congruent joint in a rabbit(Figure 1), in which the convex surface of the mandibu-lar condyle articulates with the concave articular sur-face of the temporal bone (facies articularis squamaossis temporalis) located ventrally on the zygomatic

Figure 1. Mandible of the rabbit. (a) Lateral view of the rabbit mandible. (b) Medial view of the rabbit mandible. (c) Detailof lateral aspect of the mandibular caput (arrow) (d) Detail of dorsal aspect of the mandibular caput. (e) Detail of medialaspect of the mandibular caput. (f, g) Radiographs of rabbit skull. 1: cranial aspect of the ramus mandibulae, 2: processuszygomaticus ossis temporalis, 3: processus condylaris mandibulae, 4: meatus acusticus externus, 5: bulla tympani,6: caudal aspect of the ramus mandibulae, 7: processus angularis, 8: ramus mandibulae, 9: corpus mandibualae,10: arcus zygomaticus, 11: spatium articulare of the temporomandibular joint (TMJ), 12: condylus mandibulae,13: processus caudalis arcus zygomatici, 14: os temporale, 15: os prespheonidale, w: TMJ width (TMJW).

Kyllar et al. 41

process of the temporal bone. The articular disc isformed within the joint despite the joint being congru-ent in rabbits.

Radiography

Lateral skull radiographs clearly displayed the bonystructures of the facial area. It was not possible to visu-alize the bones of the TMJ in detail due to the super-imposition of the adjacent bones and masticatorymuscles, therefore only some of the structures wereidentified (Figure 1). Radiographs gave a clear pictureof the location of the zygomatic process of the temporalbone overlying the less discernible mandibular condylefollowed by an overlap of the caudal process of thezygomatic arch (processus caudalis arcus zygomaticus).The bony part of the ear external opening and tym-panic bulla (bulla tympani) were locatedcaudally (Figure 1f).

A dorsoventral radiographic view of the rabbitskull showed the TMJ in even greater detail,allowing easy identification of the TMJ articularspace (spatium articulare), the coronoid process of themandible (processus coronoideus mandibulae), the man-dibular ramus and the wall of the temporal bone(squama ossis temporalis) with the tympanic bulla(Figure 1g).

The cross-section of the line connecting the earcanal and caudal process of the supraorbital margin(processus caudalis margo supraorbitalis) and the lineconnecting the caudal processes of the zygomatic archwas located above the joint space of the TMJ in all theskulls under investigation.

Computed tomography

All TMJ structures identified on the radiographs wereidentifiable on the CT images; however, a more detailedmorphology was visible in the transverse or sagittal CTslices (Figure 2). The articular surface of the temporalbone, which superimposed with the zygomatic arch inthe lateral view on the radiograph, was discernible onthe CT images (Figure 2). This surface was composedof a transversally elongated shallow articular tubercleof the temporal bone (tuberculum articulare ossis tem-poralis) and a shallow triangular mandibular fossa ofthe temporal bone, which was positioned sagittally andcaudally. In the dorsal view, the articular surface of themandibular condyle descended in a dorsolateral toventromedial direction. The articular surface wasalso located rostrally on the mandibular condyle inthe shape of a convex triangle, with the base situatedrostrally and the apex caudally (Figure 2). Apart froma small flat and indistinct mastoid eminence(processus mastoideus), there were no caudal borders

to the mandibular fossa. The retroarticular process(processus retroarticularis) was missing in the rabbits.Only the anterior–superior aspect of the mandibularcondyle was in contact with the articular surface ofthe temporal bone, interposing the central part of thearticular disc (Figure 2). Most of the muscular tissueswere recognizable on the CT images, including the tem-poral, masseter (musculus masseter) and pterygoid(musculus pterygoideus) muscles (Figure 2).

Magnetic resonance imaging

The soft tissues of the TMJ, including the sur-rounding muscles, were well delineated on the MRIscans. The oval articular disc was transversally pos-itioned (Figure 3). The TMJ capsule was attached tothe borders of the articular surfaces. The articular discwas connected to the medial and lateral aspects of thejoint capsule.

There are five main masticatory muscles in a rabbit(Table 1);24,27 however, only four were identified on theMR images. The masseter muscle consisted of threeheads (Figure 4). The superficial head, or pars super-ficialis, originated from a point two-thirds of the wayalong the anterior portion of the lower border of thezygomatic arch (Figure 4), and was inserted into themandibular angle (angulus mandibulae). The deephead, or pars profunda, originated one-third of theway along the medial–posterior part as well as fromthe medial portion of the zygomatic arch (Figure 4).It was inserted into the masseteric fossa (fossa masse-terica) and subdivided into a rostral and a caudal part,which were indistinct on the MR images (Figure 3).During mastication, these two heads differed infunction.

The superficial temporal muscle (musculus temporalissuperficialis) tendon slided in the muscular groove(sulcus muscularis) dorsally over the TMJ. The medialaspect of the condylar process of the mandible(processus condylaris ossis mandibularis) and the man-dibular ramus were the sites of attachment of the deeptemporal muscle (musculus temporalis profundus) andboth the pterygoid muscles (Figure 3, Table 1). Thedeep temporal muscle in the rabbits was further dividedinto the medial and lateral heads, which however werenot discernible on the MR images.

Surgical anatomy

The superficial projection of the TMJ could be locatedby palpating the osseous part of the ear canal andcaudal process of the supraorbital margin as well asthe laterally located zygomatic arch (Figure 5). Thecross-section of the axis of the zygomatic archand the line connecting the supraorbital margin and


the ear canal was located just above the TMJ space(Figure 5).

The TMJ was bordered dorsally by the zygomaticprocess of the temporal bone, medially by the squa-mous part of the temporal bone, and laterally by thezygomatic arch composed of the zygomatic process ofthe temporal bone and the temporal process of thezygomatic bone, which was projecting caudally into

the caudal process of the zygomatic arch beyond theborders of the mandibular ramus. The TMJ was caud-ally and ventrally opened and surrounded by soft tis-sues. The dorsal aspect of the TMJ was covered by themusculotendinous origin of the superficial temporalmuscle, which was directed rostrally over the zygomaticprocess of the temporal bone in the muscular groove(Figure 5) and attached rostrally from the TMJ on the

Figure 2. (a–f) Transverse and three-dimensional computed tomography images of the rabbit skull with focus on TMJ. 1:ramus mandibulae, 2: corpus mandibulae, 3: collum mandibulae, 4: arcus zygomaticus – processus temporalis ossiszygomatici, 5: arcus zygomaticus – processus zygomaticus ossis temporalis, 6: arcus zygomaticus, 7: os temporale, 8: osfrontale, 9: musculus massetericus, 10: musculus pterygoideus medialis, 11: musculus temporalis superficialis, 12:sulcus muscularis (musculus temporalis), 13: tongue, 14: condylus mandibulae, 15: condylus mandibulae, 16: porusacusticus externus, 17: processus caudalis arcus zygomatici, 18: processus zygomaticus ossis temporalis, 19: processuscondylaris, 20: margo supraorbitalis, 21: processus caudalis margo supraorbitalis, 22: processus rostralis margosupraorbitalis, 23: processus angularis mandibulae, 24: bulla tympani, 25: processus coronoideus mandibulae, 26: inci-sura supraorbitalis rostralis, 27: incisura supraorbitalis caudalis, 28: processus mastoideus.

Kyllar et al. 43

indistinct coronoid process (processus coronoideus).The TMJ was bounded medially by the deep temporalmuscle, which originated from the squamous partof the temporal bone and sphenoid bone (ossis sphenoi-dalis) and attached medially on the coronoid pro-cess and mandibular ramus. The condylar process ofthe mandible was covered caudolaterally only by thesuperficial fascia and by the skin, and was thereforeeasily palpable (Figure 5). The rostrolateral aspect ofthe zygomatic arch served as the origin of the massetermuscle – its superficial part originated from the baseof the zygomatic arch and its deep part originatedfrom the caudal process of the zygomatic arch(Figures 4 and 5).

The TMJ was surrounded by a thin capsule consist-ing of fibrous tissue and synovial lining. The capsulestretched from the edge of the mandibular fossa to theneck of the mandible (collum mandibulae) and as farcaudally as the caudal process of the zygomatic arch.The articular space was divided into superior discotem-poral and inferior discomandibular spaces. No largevessels or nerves were located in close proximity to

the rabbit TMJ. The lacrimal gland was located ros-trally and dorsally adjacent to the TMJ.

The interposed articular disc exhibited a biconcavemorphology and concave surface proximally and dis-tally (Figure 5). Its central area was thinner and the discwas attached to the joint capsule medially and laterally.

Histological analysis of the TMJ

Individual areas of the TMJ, such as the mandibularhead, mandibular fossa and fibrocartilaginous disc,exhibited differences in microscopic structure(Figure 6). The mandibular head was composed oftwo types of cartilage – hyaline cartilage and fibrocar-tilage. Collagen fibres of the fibrocartilage werearranged parallel to the surface. The fibrocartilagepassed into the hyaline cartilage in a highly cellularzone of small flattened chondrocytes, which representsthe resting zone of the cartilage. Chondrocytes in thehypertrophic zone were arranged in columns whichwere transversally orientated to the surface (Figure 6).The calcified cartilage zone was wide, especially in

Figure 3. (a–d) Magnetic resonance imaging of the rabbit head in a transversal plane with focus on TMJ. 1: ramusmandibulae, 2: condylus mandibulae, 3: processus zygomaticus ossis temporalis, 4: os temporale, 5: os preshenoidale, 6:ala ossis presphenoidalis, 7: auris externa, 8: auris media, 9: bulbus oculi, 10: discus articularis, 11: bulla tympani, 12:musculus temporalis, 13: processus pterygoideus, 14: foramen mandibulae, 15: musculus masseter, 16: musculuszygomaticomandibularis, 17: musculus pterygoideus medialis.


younger animals, and passed into the subchondral boneat a depth of about 200 mm under the surface.Trabecular bone contained a large amount of chon-dro-osseous tissue (Figure 6).

The articular disc was fibrocartilaginous (Figure 6)and revealed dense interwoven collagen Ebres orientedpredominantly mediolaterally on the periphery of thedisc, whereas the crimped collagen Ebres of the inter-mediate zone were directed rostrocaudally (Figure 6).

Morphometric assessment of rabbit TMJ

The cross-section of the line tangential to the dorsalaspect of the zygomatic arch and the line connectingthe ear canal and the caudal process of the supraorbitalmargin was located above the TMJ in all the rabbitsunder investigation. The TMJ was roughly oval inshape, with an average width of 5� 0.02mm and anaverage length of 15� 0.22mm. The width of the man-dibular condyle was 3� 0.09mm.

Morphometric analysis of the cartilage thickness inthe mandibular head area was carried out on histo-logical sections, revealing differences in cartilage thick-ness in the rostral and caudal areas. The averagethickness of the hyaline component was 196 mmrostrally, 209 mm medially and 169 mm caudally(Figure 6a,b). The thickness of the fibrous componentalso varied significantly – it was the thickest caudallyand the thinnest rostrally.

Discussion

The TMJ is unique to mammals, but its morphologyand function vary enormously among orders and spe-cies. Currently, the most commonly used model species

for studying TMJ diseases are rats, rabbits, pigs, andruminant ungulates. Each of these animal groups hasdistinctive TMJ adaptations. With the exception ofpigs, these species show less loading of the jaw jointsduring chewing than humans.28

There are morphological differences between humanand rabbit TMJs. One difference is the complete shield-ing of the rabbit TMJ by the zygomatic arch laterallyand by the condylar process of the mandible caudally inrabbits. Therefore, the entire joint is covered by theoverhanging zygomatic Fange. On the other hand, thebone arrangement on the medial side is similar to thatin humans. The most striking morphological differencebetween rabbit and human TMJs is, however, the shapeof the articular surface of the condyle and of the retro-discal area. Rabbits have no postglenoid wall. Instead,a prominent condylar process rises above the level ofthe TMJ, leaving the posterior aspect of the TMJunprotected by bone and covered only by a thin layerof temporal muscle and subcutaneous tissue. For thisreason, resection of the condylar process is necessaryfor surgical intervention in the TMJ using the posteriorapproach. That the bony shield is missing has conse-quences for posterior disc attachments, where the disc isnot rigidly anchored to the bone. In humans, the tem-poral and condylar attachments are separated by a deli-cate venous plexus, and the whole area is sheltered by apostglenoid process. This area was proposed as a spacefor disc attachments.29 The equivalent space in therabbit TMJ is Elled by the condylar process of the man-dible and the temporal muscle attached to this process.The general structure of the rabbit TMJ allows only alimited range of movements, such as hinge joint move-ments (e.g. opening and closing) and protrusive/retru-sive movements.

The anatomy and location of the rabbit TMJ makeits evaluation by radiography a challenge due to thelimited visualization of craniofacial structures. Thesuperimposition of several bones and some soft tissuesdisguises the clear pattern of the delicate structures ofthe skull. Only frontal sinuses and the facial skeletonare clearly discernible due to their superficial character.Different radiographic views have been suggested toimprove visualization of the rabbit TMJ.25,27 Anotherstudy that suggested the use of different angles of pro-jection for the visualization of the rabbit TMJ havereported a satisfactory assessment of the joint compo-nents and space in the rabbit when using a rostrocaudalview and a 10–20 degree rotation in a lateraldirection.27

Any mini-invasive treatment (injection or arthro-centesis) requiring TMJ access must always take intoaccount the surrounding anatomical structures. Inhumans, these include the ear canal, facial nerve,skull base, superficial temporal vein, maxillary artery

Figure 4. Masticatory muscle attachments in the rabbit.1: musculus temporalis superficialis, 2: musculusmasseter pars profunda caudalis, 3: musculus masseterpars superficialis (part 1), 4: musculus masseter parssuperficialis (part 2), 5: musculus masseter pars profundarostralis.

Kyllar et al. 45

and parotid glands. In rabbits, structures such as thefacial nerve, maxillary artery and parotid gland, do notneed to be considered due to the different anatomicallocation of its TMJ. To perform a mini-invasive pro-cedure in a rabbit, open surgery exposing the joint cap-sule is necessary and special care has to be taken withrespect to the joint position. Whereas the joint islocated posteriorly to the muscular prominencein humans, it is positioned anteriorly in rabbits.30

Therefore, the best approach for any mini-invasive sur-gery in a rabbit TMJ should be from a dorsal aspect ofthe TMJ. An incision should be made halfway through

the line connecting the caudal process of the supra-orbital margin and the base of the ear.

The condyle of a rabbit is covered by secondary car-tilage and fibrous tissue as in humans.31 It is alsoimportant to consider the depth of experimental defectscreated in the condylar cartilage before using the rabbitas an animal model. We proposed a depth of less than150 mm for a cartilage defect and more than 220 mm fora subchondral defect. Unlike humans, the rabbit cartil-age is thickest in the middle area and not in the poster-osuperior region. The densest part of the human TMJcartilage is 480 mm.32 This fact should be taken into

Figure 5. (a–f) Dissection of the temporomandibular joint in the rabbit. 1: margo supraorbitalis, 2: processus caudalismargo supraorbitalis, 3: ligamentum orbitale, 4: processus caudalis arcus zygomatici, 5: meatus acusticus externus,6: arcus zygomaticus, 7: line connecting processus caudalis margo supraorbitalis and meatus acusticus externus, 8: lineconnecting contralateral processus caudalis arcus zygomatici, 9: intersection of the lines defines surface projection of thetemporomandibular joint, 10: incision connecting margo supraorbitalis and meatus acusticus externus, 11: musculustemporalis, 12: capsula articularis of the temporomandibular joint, 13: processus zygomaticus ossis temporalis,14: processus condylaris mandibulae, 15: ligamentum orbitale transacted, 16: sulcus musculi temporalis, 17: condylusmandibulae/facies articularis, 18: removed processus zygomaticus ossis temporalis, 19: discus articularis, 20: cavumarticulare after the removal of the discus articularis.


Figure 6. Microscopic analysis of the rabbit temporomandibular joint (TMJ). (a–e) Sagittal sections of caput mandibulaein the rabbit. Alcian blue, haematoxylin and eosin stainings of an eight-month-old animal. (b) Graph of hyaline cartilagethickness. We observed statistically significant differences in cartilage thickness between individual areas of caputmandibulae. *P< 0.05, **0.00<P< 0.01. (c) Detail view of the cranial part of caput mandibulae. (d) Detail of the middlepart of caput mandibulae. (e) Detail of the caudal part of caput mandibulae. (f–n) Horizontal sections of articular disc. (f–h)Transitional area from disc to the capsule. (i–k) Most of the disc is formed by fibrocartilage. (l–n) Central area of the disc ismostly formed by cartilage. Scale bar in Figure A¼ 500 mm, scale bars in Figures B–N¼ 100 mm.

Kyllar et al. 47

consideration when selecting tools for the preparationof experimental defects. With regard to the arrange-ment of cartilaginous cells, there are no significant dif-ferences between human and rabbit TMJ cartilages.33

However, there are significant differences betweenhuman and rabbit TMJ discs. Whereas the humandisc is described as dense fibrous tissue, or fibrocartila-ginous plate, where the chondrocyte-like cells shouldnot be referred to as chondrocytes,34 the rabbit disccontains well-differentiated cartilaginous tissue. Thesechondrocytes are arranged between fibrous stripes, andislets of this cartilaginous tissue are present in the cen-tral area of the disc. It is worth noting that the cartil-aginous tissue is found in young animals. Nevertheless,for a deeper understanding of the origin of these cellsfurther immunohistochemical investigation will benecessary.

To identify the exact area for local injection into therabbit TMJ it is possible to use the connection of thepalpable reference points of the bony ear canal andcaudal process of the supraorbital margin and the inter-section of that line with a line tangential to the dorsalaspect of the zygomatic arch. The needle should beinserted at this point with its bevel pointing towardsand parallel with the zygomatic arch. However, thisapproach may penetrate the tendon of the temporalmuscle overlying the joint, which can cause sample con-tamination or possible tendinitis. Therefore, in cases inwhich an non-contaminated sample has to be collected,an open surgical approach above the TMJ is the onlyviable option. The incision should be performed sagit-tally above the TMJ along the line connecting thecaudal process of the supraorbital margin and thebase of the ear and by retracting the tendon of thetemporal muscle laterally. A practical execution is how-ever relatively difficult due to the size of the joint andalso to the risk of cartilage damage. Unlike in humans,it is not possible to rely on the ‘clap’ of the needle onthe fossa during its insertion. From our point of view,tilting the needle perpendicularly to the joint surfaceand performing a lateral perforation as in humansseems to be the best solution. The maximum dip ofthe needle insertion should be about 2–3mm.

An arthrocentesis in a rabbit model is obviously evenmore difficult to perform and may also require open sur-gery. Due to the differences between rabbit and humanTMJs, the rabbit TMJ is not really a suitable educa-tional model for practicing arthroscopic surgery.Rabbit TMJ can however be used as a researchmodel: while performing a surgical intervention, ana-tomical differences should be taken into account.Moreover, due to the very small size of the articulardisc, the rabbit TMJ is not suitable for performingprocedures such as arthroscopies, discopexis ordiscectomy.35



Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship, and/or publication of thisarticle: This study was supported by the Grant Agency of theCzech Republic (14-37368G to MB lab, 14-29273P to JS) and

the Ministry of Health, Czech Republic (MH CZ-DRO-FNOs/2013).

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angles. Vet J 2010; 186: 232–243.

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Oral Maxillofac Surg 2015; 44: 763–770.

Resume

L’articulation temporo-mandibulaire (ATM) est une articulation synoviale condylienne qui, avec les musclesmasticatoires, controle le mouvement mandibulaire lors de la mastication. Le lapin est souvent utilise en tantqu’espece modele pour etudier les mecanismes des maladies de l’ATM ainsi qu’en recherche regenerative. Ilexiste toutefois des differences significatives entre l’ATM du lapin et celle de l’humain, qui devraient etreprises en compte avant d’utiliser ce modele pour la recherche experimentale. Nous utilisons ici plusieursapproches analytiques (radiographie, balayage CT et IRM) pour permettre une description et une analysedetaillees de la morphologie de l’articulation temporo-mandibulaire du lapin. Des approches chirurgicalespossibles qui relient nos decouvertes a l’utilisation clinique sont egalement presentees en mettant l’accentsur l’acces disponible dans la cavite de l’ATM du lapin.

Kyllar et al. 49

Abstract

Das Kiefergelenk ist ein kondylares Synovialgelenk, dass zusammen mit Kaumuskeln dieUnterkieferbewegung beim Kauen kontrolliert. Das Kaninchen wird haufig als Modell zur Untersuchungvon Krankheiten des Kiefergelenks und in der regenerativen Forschung verwendet. Es bestehen jedochwesentliche Unterschiede zwischen dem Kiefergelenk von Kaninchen und Mensch, die vor einer Nutzungdieses Modells fur experimentelle Forschung berucksichtigt werden mussen. Anhand verschiedenerAnalyseverfahren (Radiografie, CT und MRI) ermoglichen wir hier eine detaillierte Beschreibung undAnalyse der Kiefergelenkmorphologie des Kaninchens. Zudem werden mogliche chirurgische Verfahrenmit Schwerpunkt auf verfugbarem Zugang zur Kiefergelenkhohle des Kaninchens vorgestellt, die unsereErkenntnisse mit klinischem Einsatz verbinden.

Resumen

La articulacion temporomandibular (TMJ) es una articulacion sinovial condilar que, junto con los musculosmasticatorios, controla el movimiento durante la masticacion. El conejo suele utilizarse como especie modelopara estudiar los mecanismos de los trastornos de TMJ y para la investigacion regenerativa. No obstante,existen diferencias significativas entre la TMJ en humanos y en conejos que deberıan tenerse en cuenta antesde utilizar este modelo para la investigacion experimental. En este estudio utilizamos varios metodosanalıticos (radiografıa, CT y MRI) para conseguir una descripcion y un analisis precisos de la morfologıa dela articulacion temporomandibular. Asimismo, se introducen metodos quirurgicos posibles con un foco en elacceso disponible a la cavidad de TMJ del conejo, uniendo de este modo nuestras conclusiones al uso clınico.


ab oratory

l i m i t e d

lan imals

Original Article

Sedative and cardiorespiratory effects ofdetomidine constant rate infusion in sheep

Rauane Sousa de Moura, Isabela Plazza Bittar,Luiz Henrique da Silva, Ana Carolina Vasquez Villela,Marcelo Borges dos Santos Junior, Naida Cristina Borgesand Leandro Guimaraes Franco

AbstractThe use of sheep in experiments is widespread and is increasing worldwide, and so is the need to developspecies-specific anaesthetic techniques to ensure animal safety. Previous studies have mentioned severalprotocols involving the administration of alpha-2 adrenergic agonists in sheep; however, assessment of theefficacy and safety of these infusion techniques is still relatively new. Thus, the aim of the present study is toassess the effectiveness of detomidine constant rate infusion (CRI) in sheep by measuring the cardiovascularand respiratory parameters, blood gas variables and sedation scores. Eight adult female Santa Ines sheepreceived 20 mg/kg of detomidine hydrochloride intravenously as a bolus loading dose, followed by an infusionrate of 60 mg/kg/h. The heart rates and respiratory rates changed continuously during the CRI period. Noarrhythmias were observed. The reduction in arterial partial pressure of oxygen (PaO2) was not significant, butone animal showed signs of hypoxaemia (minimum PaO2 of 66.9mmHg). The arterial partial pressure ofcarbon dioxide (PaCO2) increased, but the animals did not become hypercapnic. The bicarbonate (HCO3�),pH and base excess (BE) tended towards metabolic alkalosis. The cardiac output (CO), stroke volume (SV),cardiac index (CI) and ejection fraction (EF%) showed no significant changes. The fractional shortening (FS%)decreased slightly, starting at T45min. Sedation scores varied between 3 (0/10) after sedation and duringrecovery and 7 (0/10) during CRI. We concluded that administering detomidine at an infusion rate of60 mg/kg/h in Santa Ines sheep is a simple technique that produces satisfactory sedation for minimally inva-sive procedures.

Keywordsconstant rate infusion, alpha-2 adrenergic agonists, small ruminants, experimental models

Date received: 5 May 2016; accepted: 14 May 2017

The sheep is a convenient and popular large animalmodel for biomedical research. The need to determinespecies-specific sedation protocols has increased signifi-cantly, as most drugs used in sheep are often selectedbased on their effectiveness in other species. Ideal proto-cols for chemical restraint and sedation should facilitateanimal handling, systemic analgesia and rapid recov-ery.1 The development of adequate sedative and anal-gesic protocols should focus on simplifying techniques,on causing no detrimental side-effects, and on minimiz-ing general anaesthesia requirements for surgical pro-cedures, thus reducing associated complications.2,3

Alpha-2 adrenergic agonists are potent sedativedrugs that provide muscle relaxation and analgesia

and are widely used in sheep.1–5 Although this classof drugs is widely used, dose-dependent cardiopulmon-ary effects, such as decreased cardiac output (CO), bra-dyarrhythmia, peripheral vasoconstriction, biphasicarterial blood pressure response, respiratory

Department of Veterinary Medicine, Federal University of Goias,Brazil

Corresponding author:Leandro Guimaraes Franco, Department of Veterinary Medicine,School of Veterinary Medicine and Animal Science, FederalUniversity of Goias, Campus Samambaia, Av Esperanca s/n, CEP74690-900, Goiania-GO, Brazil.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 51–58



sagepub.co.uk/


DOI: 10.1177/0023677217714013


depression, hypoxaemia and acute pulmonary oedema,have been observed.6–11 Also, individual, breed orstrain variability can influence the presence or absenceof such effects.7

Furthermore, ruminants are known to be more sen-sitive to the effects of alpha-2 adrenergic agonists, whencompared with other species, such as horses.1,3

Detomidine is more alpha-2-specific than xylazine,which means that a lower dose is required to achievea desirable sedation with fewer side-effects.12

Administration routes for detomidine include intra-muscular (IM) and intravenous (IV) as a bolus or con-stant rate infusion (CRI).13 Cardiopulmonary effectshave a faster onset in IV routes than IM routes,which tend to be more reliable.1,2,5,14

In other species, such as horses, the administrationof IV detomidine as a bolus3,5,7,15 or CRI15–18 is wellestablished, and has been proven to cause excellent sed-ation without significant cardiopulmonary depressionand ataxia.3,16,19 For standing chemical restraint, infu-sion rates varying from 5 to 40 mg/kg/h have beenproven to be effective for prolonged sedation, withthe caveat that they do not provide sufficient analgesiafor surgical procedures.16–18 Other studies have com-pared steady state infusion with IV bolus and con-cluded that the plasma concentration of detomidine isdirectly proportional to adequate sedation and cardio-vascular responses.15

In sheep, a xylazine infusion regimen provided aneffective and predictable steady state analgesia withoutthe variability observed with bolus administration.8 Inpregnant ewes and preterm foetal sheep, the infusion ofdexmedetomidine produced satisfactory results for sed-ation and pain control, with minimal foetal response.10

These studies did not evaluate the occurrence of hypox-aemia or other respiratory effects. However, the effectsof detomidine CRI are yet to be reported. It is believedthat this technique can also provide prolonged and goodquality sedation with minimal side-effects.15 Moreover,there is not enough information regarding the haemo-dynamic parameters, sedation scores or infusion dosesfor sheep. Thus, the present study assesses the effective-ness and safety of detomidine CRI in sheep through theevaluation of cardiovascular and respiratory param-eters, blood gas variables and sedation scores.


Animals

The experimental protocol was approved by the EthicsCommittee for Animal Research of Goias FederalUniversity (UFG), Brazil. Eight clinically healthyadult female Santa Ines sheep (Fazenda Anicuns,Anapolis, Brazil), weighing 34.98� 6.32 kg

(mean� standard deviation [SD]), were used. The ani-mals were kept indoors, fed with alfalfa hay and com-mercial food (Nutroeste Nutricao Animal, Goiania,Brazil) twice daily, and provided with water ad libitum.

Experimental preparation

The animals were housed in group pens in the researchfacility with enough time to acclimatize to the environ-ment. Feed was withheld for 18 h and water for 4 hprior to the experimental procedures. For drug admin-istration, a 16G intravascular catheter (Angiocath;Becton-Dickinson Infusion Therapy Systems Inc,Sandy, UT, USA) was inserted into the left jugularvein. For invasive blood pressure assessment andblood gas analysis, a 22G intravascular catheter(Angiocath) was inserted into the auricular artery andkept patent with a heparin lock.

Drug treatment

Detomidine hydrochloride 20 mg/kg (Dormiun V;Agener Uniao–Saude Animal, Sao Paulo, Brazil) wasgiven intravenously as a bolus loading dose followed byan infusion rate of 60 mg/kg/h. The interval between thebolus dose and the onset of infusion was 10min, afterwhich the sedation scores were assessed. The sheep wereplaced in right lateral recumbency, and the infusion wasadministered using an automatic syringe pump(DigiPump SR8x; Digicare Animal Health, BoyntonBeach, FL, USA). During the experimental anaestheticprocedure, the animals were spontaneously breathingroom air, and 0.9% saline was administered at a main-tenance rate of 5mL/kg/h. The detomidine CRI periodwas 60min.

Assessments

Heart rate and rhythm; respiratory rate; mean, systolicand diastolic blood pressures; and peripheral oxygensaturation (SpO2) were evaluated using a multi-para-meter monitor (Dixtal DX2010; Dixtal BiomedicaIndustria e Comercio Ltda, Manaus AM, Brazil). Thearterial partial pressure of oxygen (PaO2), arterial par-tial pressure of carbon dioxide (PaCO2), bicarbonate(HCO3�), base excess (BE), and pH were evaluatedusing a blood gas and electrolyte analyser (Cobas b121; Hoffmann-La Roche Ltd, Basel, Switzerland).Sedation was assessed by a single experienced observer,based on the Kastner et al. (2003)20 study, whichassigned scores from 0 to 10, where 0 represented stand-ing, with alert and normal behaviour, and 10 repre-sented lateral recumbency, with no movements.

In addition, the cardiovascular function qualitativeanalysis was performed using Doppler echocardiography


in two-dimensional mode through the right parasternalwindow. The obtained images allowed the measurementof interventricular septal thickness in systole (IVSs) anddiastole (IVSd); left ventricular diameter in systole(LVIDs) and diastole (LVIDd); and left ventricularfree wall thickness in systole (LVFWs) and diastole(LVFWd); followed by calculation of left ventricularfractional shortening (FS%) and ejection fraction(EF%). The cardiac index (CI) was calculated basedon CO, using the following formula: CO/body surfacearea (BSA), where BSA¼weight0,667/10. To determineCO, images were obtained from the right ventricularoutflow tract, in which the annulus diameter of the pul-monary valve was measured, and pulmonary artery flowwas subsequently recorded to determine the heart rate.The images were obtained using an ultrasound machine(Logic-e ultrasound – BT12; GE Healthcare, LittleChalfont, UK).

All parameters were evaluated before the experimen-tal procedure (TBASAL), and the baseline values weredetermined as the arithmetic means of three consecutivemeasurements. All parameters were then evaluated atthe following times: at the start of detomidine CRI(10min after loading the bolus) (T0), every 15minduring infusion (T15, T30, T45, T60) and 30min afterthe end of CRI (TREC). At all evaluation times, thesheep were observed for indications of sedation, includ-ing increases in salivation and degree of ptosis, and

changes in responsiveness to external non-painful sti-muli, such as noise. All voluntary attempts to movewere recorded.

Data analysis

The data were summarized and expressed as themean�SD. Statistical analyses were performed bySigmaPlot for Windows (version 11.0). The Shapiro–Wilk test was used to verify a normal data distribution.Parametric values were analysed by one-way repeatedmeasures analysis of variance (ANOVA), followed byTukey’s test. Friedman’s test was chosen for non-parametric data. Values of P< 0.05 were consideredto be significant.

Results

The sedation scores (median and quartiles) recordedduring detomidine CRI are shown in Figure 1. Themean values and SDs of clinical parameters evaluatedduring the experimental procedure are summarized inTable 1.

The heart rate decreased continuously, starting at T15,and the sheep became bradycardic (heart rate below 80beats/min)21 30min after infusion started, but noarrhythmias were observed. The respiratory ratedecreased at T30, but remained within normal reference

Figure 1. Sedation scores (median� quartiles) in response to 20 mg/kg of intravenous detomidine bolus followed by acontinuous infusion of 60 mg/kg/h.TBASAL: time of bolus injection; T0: time when infusion started; TREC: time at recovery.

Sousa de Moura et al. 53

values for the species. The values for both these variablesreturned to baseline at recovery (TREC). Blood pressurealso remained within normal reference values during the90min evaluation period; neither hypertension (systolic,diastolic and mean blood pressure above 130, 90 and110mmHg, respectively)22 nor hypotension (systolic,diastolic and mean blood pressure below 90, 60 and70mmHg, respectively)22 was observed. The body tem-perature decreased gradually, and the lowest values wereobtained after 45min of infusion.

Results from the arterial blood gas analysis showed nosignificant changes in PaO2 mean values. One animalshowed a significant decrease in PaO2 at T30, comparedwith baseline (TBASAL¼ 73.3mmHg; T30¼ 68.9mmHg).Another animal remained significantly hypoxaemic(PaO2 below 80mmHg)23 during the whole infusionperiod (TBASAL¼ 81.7mmHg; T0¼ 69.6mmHg;T60¼ 67.4mmHg). Both returned to values similar tobaseline at T45 and TREC, respectively, while still in lateralrecumbency. A significant increase in the PaCO2 meanvalues occurred gradually in all animals. The HCO3�,pH and BE values increased, starting at T30.

The cardiovascular function variables remained con-stant across the experimental period. Parameters suchas CI, EF% and stroke volume (SV), were not alteredsignificantly, compared with the baseline values.However, FS% showed a significant decrease at T45

and T60.

Regarding the sedation scores, the animals receiveda grade of 3 (0/10) at T0, and this increased to 7 (0/10)at T15. Throughout the infusion period (T30, T45, T60),the sedation scores remained constant (7/10). At TREC,during the anaesthetic recovery period, the animals’average grade was 3, similar to T0.

Discussion

Despite previous reports of complications after theadministration of alpha-2 adrenergic agonists insheep, neither arrhythmias nor breathing patternchanges were observed in the present study. However,decreases in heart and respiratory rates were alsoexpected, due to the inhibition of sympathetic tonecaused by presynaptic norepinephrine reuptake.Bradycardia is a common side-effect, which was con-firmed by our results.6,24 In previous studies, similarresults for heart rates were reported with IV adminis-tration of detomidine 30 mg/kg in sheep7 and 10 mg/kg incattle.14 Contrary to our results, Celly et al. reported asignificant increase in the respiratory rate after detomi-dine administration, starting 2min after the bolus andlasting up to 30min. In the present study, no assess-ments were conducted in the first 10min after the load-ing dose; however, no significant changes were observedat T0 and T15, compared with baseline. At T30 and T60,the respiratory rate decreased significantly.

Table 1. Clinical parameters in response to 20 mg/kg bolus of intravenous detomidine followed by continuous adminis-tration of 60 mg/kg/h in domestic sheep (n¼ 8).

TBASAL T0 T15 T30 T45 T60 TREC

HR 105� 17 61� 7 57� 10* 53� 9* 52� 8* 55� 10* 79� 25*

RR 31� 6 26� 7 27� 7 25� 8* 26� 9 25� 9* 26� 5

SpO2 95� 1 93� 3 93� 3 92� 1 93� 3 93� 2 93� 3

SBP 119� 16 101� 20 96� 10 110� 9 111� 14 109� 15 90� 33

DBP 96� 12 86� 19 76� 26 94� 8 95� 10 96� 10 89� 11

MAP 109� 11 94� 18 93� 10 102� 7 102� 11 103� 10 96� 11

T�C 39.3� 0.3 39.2� 0.3 38.9� 0.5 38.7� 0.3* 38.3� 0.5* 37.9� 0.6* 37.3� 0.5*

CI 2.09� 0.63 2.03� 0.53 1.91� 0.54 1.50� 0.54 1.68� 0.66 1.86� 0.38 1.98� 0.72

EF% 73.0� 9.4 67.4� 9.0 67.9� 12.8 65.2� 11.8 60.7� 8.3 58.2� 9.7 63.4� 11.6

FS% 45.3� 8.5 36.8� 6.8 37.8� 9.5 35.5� 9.0 31.8� 5.8* 30.3� 6.7* 34.0� 8.1

SV 28.5� 5.5 27.2� 6.5 27.9� 6.5 25.6� 3.2 25.7� 4.4 28.1� 6.9 26.2� 8.2

pH 7.46� 0.02 7.47� 0.01 7.48� 0.01 7.48� 0.01 7.49� 0.02* 7.48� 0.02 7.49� 0.02*

PaO2 79.1� 3.5 74.6� 4.3 74.5� 6.5 77.0� 3.7 79.2� 5.5 81.0� 6.9 83.3� 5.9

PaCO2 33.2� 2.7 35.5� 2.3* 37.3� 2.3* 37.8� 2.5* 38.1� 3.1* 39.6� 3.1* 39.9� 3.4*

HCO3� 24.6� 3.6 26.6� 3.3 28.3� 3.5 29.1� 3.5* 29.4� 2.6* 30.4� 3.1* 31.3� 2.7*

BE 0.1� 1.4 2.1� 1.3 3.7� 1.7 4.6� 1.7* 5.3� 1.8* 5.8� 1.9* 6.6� 1.6*

HR: heart rate; RR: respiratory rate; SpO2: peripheral oxygen saturation; SBP: systolic blood pressure; DBP: diastolic blood pressure;MAP: mean arterial pressure; T�C: temperature (degrees Celsius); CI: cardiac index; EF%: ejection fraction; FS%: fractional shortening;SV: stroke volume; PaO2: arterial partial pressure of oxygen; PaCO2: arterial partial pressure of carbon dioxide; HCO3�: bicarbonate; BE:base excess; TBASAL: time of bolus injection; T0: time when infusion started; TREC: time at recovery. Values are given as mean� standarddeviation (*P< 0.05).


A biphasic arterial blood pressure response was alsoexpected after the administration of alpha-2 adrenergicagonist.7,14,25,26 Due to the initial stimulation of alpha-1 adrenergic receptors, this blood pressure response ischaracterized by transient hypertension followed byhypotension1 and is responsible for peripheral vasocon-striction.6 No significant changes in the mean bloodpressure values were observed during the experimentalperiod. However, because evaluation began only 10minafter the detomidine loading dose, it is possible that thetransient hypertensive peak was missed.

In the present study, no significant changes in PaO2

mean values were observed. On the other hand, a sig-nificant increase in PaCO2 was observed, probably dueto a decrease in ventilation which led to a CO2 build-up. Alpha-2 adrenergic agonists have been reported toinduce a variable degree of hypoxaemia in sheep afterIV administration.7,9,11,20 So, despite the resultsobtained, it is worth considering individual, breed andstrain variabilities. Two animals (2/8) showed signifi-cant reductions in PaO2 values, which confirm theeffects of alpha-2 adrenergic agonists on oxygenation.Our results showed a reduced occurrence of hypox-aemia, but it is not possible to infer that these resultswould be similar under other conditions such as bodyposition or breed selection. Thus, the establishment ofoxygen therapy is strongly recommended, depending onthe individual response. Hypoxaemia can be attributedeither to hypoventilation or the presence of pulmonaryoedema; however due to the limitations of our study,we could not determine why only two animals showedsevere hypoxaemia (PaO2 below 70mmHg).23

The variables HCO3�, pH and BE increased grad-ually, and values obtained for pH and HCO3� in sheepwere similar to those observed in horses in response to adetomidine infusion rate of 20 mg/kg/h.15 Although thevalues also remained within the normal reference valuesfor the species, there was a tendency toward metabolicalkalosis. Thus, based on blood gas analysis, it can beinferred that continuous detomidine administrationmay further lead to metabolic alkalosis as a compensa-tion for respiratory acidosis.7

Cardiovascular function parameters remained con-stant across the experimental period; however, FS%values had decreased at two of the evaluation stages(T45 and T60). A decrease in FS% alone does not dir-ectly interfere with left ventricular systolic function as itcan be influenced by preload and afterload, which werenot evaluated in this study. Furthermore, the standardparameter for systolic function evaluation is EF%,which remained constant.27 Alpha-2 adrenergic agon-ists are expected to decrease the CI values,24,28,29

mainly due to a decreased heart rate.24,28 Although asignificant decrease in heart rate took place, there wereno changes in CI values, unlike the results of previous

studies.24,30 The present results were obtained by mea-suring CO using echocardiography; and although reli-able values were obtained from aortic and pulmonaryarterial flow in other species,27,31,32 these values havebeen proven to be highly correlated with the thermo-dilution technique,33 and high variability had previ-ously been observed in sheep.28

In this study sedation scores varying between 3 (0/10)after IV detomidine bolus and 7 (0/10) during CRI wereconsidered to be satisfactory for performing simple pro-cedures that were minimally invasive and not painful.During CRI, the sheep remained in lateral recumbencyand were unresponsive to noise, however after the touch-ing stimuli, they assumed sternal recumbency but werestill unable to support their heads. External stimuli werenot painful and consisted of encouraging the sheep tochange position by touching them. Similar depths of sed-ation were obtained after IV administration of 50mg/kgdetomidine in goats2 and 40mg/kg in sheep. However, thesedation patterns were more variable, achieving optimalsedation periods with subsequent decreases, unlike thoseobservedwithCRI.5 In addition, the animals showed pro-longed recovery times, between 605 and 120min,2 whichwere also different from the values obtained in the presentstudy. The observed recovery can be explained by thepreviously proven correlation between sedation anddrug plasma concentration.20

We can conclude that detomidine infusion at a rateof 60 mg/kg/h in Santa Ines sheep is a simple techniquethat can produce satisfactory sedation to facilitatehandling and be used for minimally invasive proced-ures. The sedation produces no significant cardio-respiratory effects and provides rapid and high qualityrecovery. Despite these promising results, it is worthconsidering individual variability, and it is also worthevaluating the need to establish oxygen therapy in theevent of hypoxaemia.

Acknowledgements

We thank Programa de Pos-Graduacao em Ciencia Animal

from the Federal University of Goias (UFG) for the award ofa Master’s scholarship grant, and the Veterinary TeachingHospital where the experiment was conducted.



Funding

The author(s) disclosed receipt of the following financial sup-port for the research, authorship, and/or publication of thisarticle: A Master’s scholarship grant was awarded by the

Programa de Pos-Graduacao em Ciencia Animal from theFederal University of Goias (UFG).


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Resume

La popularite de l’experimentation sur les moutons augmente dans le monde entier, de meme que la neces-site de developper des techniques d’anesthesie specifiques aux especes pour assurer la securite des ani-maux. Des etudes precedentes mentionnent plusieurs protocoles impliquant l’administration d’agonistesadrenergiques alpha-2 chez les moutons. Cependant, l’evaluation de l’efficacite et de la securite de cestechniques de perfusion est encore relativement nouvelle. Ainsi, l’objectif de la presente etude est d’evaluerl’efficacite de la perfusion a debit constant de detomidine (CRI) chez les moutons en mesurant les parametrescardiovasculaires et respiratoires, les variables de gaz dans le sang et les scores de sedation. Huit femellesadultes Santa Ines ont recu 20 mg/kg de chlorhydrate de detomidine par voie intraveineuse en tant que dose decharge bolus, suivie d’un taux de perfusion de 60 mg/kg/h. Les frequences cardiaques et respiratoires ontchange de facon continue pendant la periode de CRI. Aucune arythmie n’a ete observee. La reduction de lapression partielle arterielle en oxygene (PaO2) n’etait pas significative, mais un animal presentait des signesd’hypoxemie (PaO2 minimum de 66,9mmHg). La pression partielle arterielle en dioxyde de carbone (PaCO2) aaugmente, mais les animaux ne sont pas devenus hypercapniques. Le bicarbonate (HCO3-), le pH et l’exces debase (BE) tendaient a l’alcalose metabolique. Le debit cardiaque (CO), le volume d’ejection systolique (SV),l’index cardiaque (IC) et la fraction d’ejection (EF%) n’ont pas montre de changements significatifs. La fractionde raccourcissement (FS%) a legerement diminue, a 45 minutes. Les scores de sedation variaient entre 3 (0/10) apres la sedation et pendant le retablissement et 7 (0/10) pendant la CRI. Nous avons conclu que l’admin-istration de detomidine a un debit de perfusion de 60 mg/kg/h chez le mouton de Santa Ines s’avere etre unetechnique simple qui produit une sedation satisfaisante pour des procedures peu invasives

Abstract

Da weltweit immer mehr mit Schafen experimentiert wird, besteht erhohter Bedarf an der Entwicklung arten-spezifischer Narkosemethoden, um die Sicherheit der Tiere zu gewahrleisten. Zwar wurden in fruherenStudien verschiedene Protokolle zur Verabreichung von adrenergischen Alpha-2-Antagonisten an Schafeerwahnt, die Bewertung der Wirksamkeit und Sicherheit dieser Infusionstechniken steht jedoch noch relativam Anfang. Ziel der vorliegenden Studie war daher die Bewertung der Wirksamkeit von konstanterDetomidin-Infusion (CRI) bei Schafen durch Messung der kardiovaskularen und respiratorischenParameter, Blutgas- und Sedierungswerte. Acht weibliche Santa Ines-Schafe erhielten 20 mg/kgDetomidinhydrochlorid intravenos als Bolus-Ladedosis, gefolgt von einer Infusionsrate von 60 mg/kg/h. DieHerz- und Atemfrequenzen anderten sich wahrend der CRI-Periode kontinuierlich. Es wurden keineArrhythmien beobachtet. Die Senkung des arteriellen Sauerstoffpartialdrucks (PaO2) war unerheblich, dochein Tier zeigte Anzeichen von Hypoxamie (Minimum PaO2 von 66,9mmHg). Der arterielleKohlendioxidpartialdruck (PaCO2) stieg, aber die Tiere zeigten keine Hyperkapnie. Bikarbonat (HCO3-), pHund Basenuberschuss (BE) tendierten zu metabolischer Alkalose. Herzzeitvolumen (CO), Schlagvolumen (SV),Herzindex (CI) und Ejektionsfraktion (EF%) zeigten keine wesentlichen Anderungen. Fractional Shortening(FS%) sank leicht, ab T45min. Sedierungswerte variierten zwischen 3 (0/10) nach Sedierung und wahrend derAufwachphase und 7 (0/10) wahrend der CRI. Wir schlussfolgerten, dass Detomidin- Verabreichung mittels


einer Infusionsrate von 60 mg/kg/h bei Santa Ines-Schafen eine einfache Methode zur ausreichendenSedierung bei minimal invasiven Verfahren darstellt.

Resumen

La popularidad de experimentar con ovejas esta aumentando en todo el mundo, y tambien lo esta haciendo lanecesidad de desarrollar tecnicas de anestesia especıficas para cada especie con el fin de garantizar laseguridad del animal. Estudios anteriores mencionan varios protocolos sobre la administracion de agonistasadrenergicos alfa2 en ovejas; sin embargo, la evaluacion de la eficacia y seguridad de estas tecnicas deinfusion es todavıa relativamente nueva. Por tanto, el objetivo del presente estudio es evaluar la eficaciade la infusion a un ritmo constante de detomidina (CRI) en ovejas midiendo los parametros cardiovasculares yrespiratorios, las variables de los gases sanguıneos y los marcadores de sedacion. Ocho ovejas Santa Inesadultas hembra recibieron 20 mg/kg de hidrocloruro de detomidina de forma intravenosa como una dosis decarga bolo, seguido de un ritmo de infusion de 60 mg/kg/h. Los ritmos cardıacos y respiratorios cambiaroncontinuamente durante el periodo de CRI. No se observo ninguna arritmia. La reduccion de la presion parcialarterial de oxıgeno (PaO2) no fue significativa, pero un animal mostro signos de hipoxemia (mınimo PaO2 de66,9mmHg). La presion parcial arterial de dioxido de carbono (PaCO2) aumento pero los animales no pre-sentaron ningun problema hipercapnico. El bicarbonato (HCO3-), pH y el exceso de base (BE) se inclinaronhacia la alcalosis metabolica. El gasto cardıaco (CO), el volumen de bombeo (SV), el ındice cardıaco (CI) y lafraccion de expulsion (EF%) no mostraron cambios significativos. El recorte fraccional (FS%) disminuyoligeramente, empezando en T45min. Los ındices de sedacion oscilaron entre 3 (0/10) despues de la sedaciony durante la recuperacion y 7 (0/10) durante CRI. Concluimos que la administracion de detomidina a un ritmode infusion de 60 mg/kg/h en ovejas Santa Ines es una tecnica sencilla que produce una sedacion satisfactoriapara procedimientos invasivos mınimos.


ab oratory

l i m i t e d

lan imals

Original Article

TNFa depleting therapy improves fertilityand animal welfare in TNFa-driventransgenic models of polyarthritis whenadministered in their routine breeding

Amy J. Naylor1, Guillaume Desanti1, Atif N. Saghir1 andRowan S. Hardy1,2,3

AbstractTransgenic tumour necrosis factor alpha (TNFa)-driven models of polyarthritis such as the TNF�ARE mousehave proven to be invaluable in delineating aspects of inflammatory disease pathophysiology in humans.Unfortunately, the onset of joint destruction and inflammation in these models represents a significant det-riment to breeding management. We examined whether TNFa depleting therapy ‘infliximab’ might represent asignificant refinement in routine breeding. Clinical scores of joint inflammation were assessed in TNF�ARE

males receiving either infliximab (10mg/kg) or saline by twice-weekly intraperitoneal injection. Joint histologyand bone morphology were assessed by histological analysis and micro-computed tomography (CT), respect-ively. Analysis of breeding was examined retrospectively in TNF�ARE males prior to, and following, regularintroduction of infliximab. Clinical scores of inflammation were significantly reduced in TNF�ARE malesreceiving infliximab (control 6.6 arbitrary units [AU]� 0.88 versus infliximab 4.4 AU� 1.4; P< 0.05), whilemeasures of pannus invasion and bone erosion by histology and micro-CT were markedly reduced. In thebreeding groups, TNF�ARE males receiving infliximab injections sired more litters over their breeding lifespan(control 1.69� 0.22 versus infliximab 3.00� 0.19; P< 0.005). Furthermore, prior to infliximab, TNF�ARE maleshad a 26% risk of failing to sire any litters. This was reduced to 7% after the introduction of infliximab. Thisstudy is the first to report that regular administration of infliximab is effective at suppressing disease activityand improving animal welfare in TNF�ARE animals. In addition, we have shown that infliximab is highly effi-cacious in improving breeding behaviour and increasing the number of litters sired by TNF�ARE males.

Keywordsmurine polyarthritis, breeding, infliximab, refinement

Date received: 10 March 2017; accepted: 11 April 2017

Rheumatoid arthritis (RA) is a chronic auto-immune inflammatory disease resulting in polyarticularinflammation, joint destruction and systemicinflammatory complications. Murine models of polyar-thritis closely model features of RA in humans andhave proven to be effective in advancing our under-standing of the pathophysiology of inflammatorydisease.

The primacy of murine models of inflammatory dis-ease is underpinned by several key factors: parallelsbetween the immune responses, their highly conservedgenetic background, high progeny numbers, and shortbreeding times that have enabled their extensive useand validation in medical research.1

A range of transgenic murine models of spontaneouspolyarthritis exist, including the hTNFtg, TNF�ARE,KBxN, SKG and DNase II mice.2–6 These models

1Institute of Inflammation and Ageing, University of Birmingham,Birmingham, UK2Institute of Metabolism and Systems Research, University ofBirmingham, Birmingham, UK3Centre for Endocrinology Diabetes and Metabolism, BirminghamHealth Partners, Edgbaston, Birmingham, UK

Corresponding author:R S Hardy, PgC, PhD, Institute of Inflammation and Ageing,University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 59–68



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DOI: 10.1177/0023677217707985


offer several advantages relative to inducible models,the major benefits being the strong parallels withchronic inflammatory disease in humans and develop-ment of polyarthritis in a highly reproducible and tem-porally-controlled manner, making them ideal forexamining therapeutic disease interventions. Indeed,in the seminal study by Keffer et al., a once-weeklyinjection of infliximab completely abrogated diseaseactivity in hTNFtg mice, contributing to the now com-monplace application of biological anti-tumour necro-sis factor alpha (TNFa) therapies as a gold standard inthe treatment of chronic inflammatory disease.2,7,8

The hTNFtg model (known as Tg(TNF)197Gkl)possesses an insertion of a 30-modified human TNFtransgene which greatly increases TNFa mRNA stabil-ity resulting in systemic overproduction of humanTNFa.2 In the TNF�ARE mouse, the 30 untranslatedregion (UTR) of the AU-rich element (ARE) in themurine TNFa gene is deleted, resulting in greatlyincreased TNFa mRNA stability and systemic overex-pression.6 As a consequence, inflammation is driven bythe overexpression of TNFa in all major tissues result-ing in polyarthritis and joint destruction in bothmodels. In addition, the TNF�ARE mouse presentswith an inflammatory intestinal pathology that closelymodels inflammatory bowel disease as well as occa-sional mild inflammation in the liver and the lung.These models have helped in delineating the patho-physiology of inflammatory diseases such as RA inhumans, demonstrating the role that TNFa plays atthe apex of a pro-inflammatory cytokine cascade, med-iating leukocyte infiltration, synovitis and jointdestruction.9

One notable drawback associated with the use oftransgenic models of polyarthritis occurs in maintain-ing breeding colonies required to generate experimentalanimals. Both hTNFtg and TNF�ARE mice are bredusing a heterozygous transgenic male crossed with awild-type female to generate offspring with a 50% inci-dence of developing spontaneous polyarthritis. Whilerequired in experimental animals, the spontaneousonset of polyarthritis and systemic inflammation inbreeding males is of significant detriment to their wel-fare and breeding success.10

From 2–4 weeks of age, heterozygous TNF�ARE

mice present with early signs of inflammatory lesionswithin the terminal ileum and proximal colon andbecome more severe around eight weeks. The histo-logical and clinical onset of polyarthritis is detectedbetween 6 and 8 weeks in TNF�ARE mice and becomesincreasingly severe up to 14 weeks. Following onset ofarthritis, animals are scored for joint inflammation, aswell as behaviour, mobility, weight loss and grimace.6

A number of welfare measures, as outlined by Hawkinset al., are observed in the maintenance of these

transgenic animals to mitigate these factors andenhance breeding. These include soft litter to reducepain on walking, non-tangling nesting material andeffortless access to food and water to cater fordisability. Lastly the introduction of opiate pain reliefis utilized where arthritis is evident to minimize pain anddistress, given once daily at first onset (0.1mg/kg sub-cutaneous buprenorphine).11,12 Ultimately, the intro-duction of clearly defined humane endpoints forarticular inflammation scores (supplementary Table 1,see http://lan.sagepub.com for all supplementary mater-ial), and clinical scores of weight loss, behaviour, mobil-ity, grimace, severity and duration of joint inflammation(supplementary Table 2), ensure that breeding males donot experience excessive distress or pain. Unfortunately,this reduces their breeding lifespan to around 14–18weeks, relative to 35–40 weeks in wild-type animals.

Infliximab is a neutralizing and depleting antibodytargeted at human TNFa, and is successfully used toameliorate joint destruction and inflammation inhTNFtg mice.13,14 Interestingly, while infliximab is notreported to have cross reactivity with murine TNFa(which drives polyarthritis in the TNF�ARE model),the reported suppression of inflammation and TNFain murine models of colitis and diabetes suggest furthermodes of action.15–19 Therefore, given the central role ofTNFa in the pathophysiology of the TNF�ARE model,we examined whether a similar regimen of regular inflix-imab injections would improve management of our het-erozygous TNF�ARE breeding programmes.

Consequently, in this study we show that regularadministration of infliximab to breeding TNF�ARE

males provided a significant refinement in the breedingprogramme, improving animal welfare by suppressingdisease activity and reducing the numbers of breedinganimals required to generate experimental TNF�ARE

animals.


Animals

C57BL/6 TNF�ARE mice were obtained fromDr George Kollias (BSRC Fleming, Athens, Greece).6

At 6–8 weeks, heterozygous TNF�ARE male mice(TNF�ARE/þ, later referred as TNF�ARE) developedclinical signs of systemic polyarthritis, with 100% pene-trance. The mice were housed under controlled envir-onmental conditions (20.2� 2�C, 14:10 h light:darkcycle). The experiments were carried out followingstrict guidelines governed by the UK Animal(Scientific Procedures) Act 1986 and approved by theBirmingham Ethical Review Subcommittee. Only maleanimals were utilized for the examination of polyarthri-tis and breeding.


TNFARE transgenic mouse model andclinical scoring

Preparations of infliximab (Remicade�; JanssenBiotech, Horsham, PA, USA) were reconstituted at10mg/mL in sterile water according to the manufac-turer’s guidelines. To assess the effects of infliximabon disease activity, TNF�ARE males from six weeksof age were given either 10mg/kg of infliximab orsaline (100 mL of 0.9% NaCl in distilled water) bytwice-weekly intraperitoneal injection. For scoringjoint inflammation and polyarthritis, infliximab injec-tions were split to ensure that within any one litter, onemale TNF�ARE sibling would receive infliximab andone would receive the saline control. Animals werethen scored separately in a blinded manner.TNF�ARE animals were caught by their tail, liftedwith hand support and immediately transferred ontoa metallic cage to minimize involuntary extension/flex-ion of arthritic joints. They were then restrained by theback and lifted to perform paw monitoring. The ani-mals were familiarized with handling and observationfor one week prior to the onset of experiments to min-imize stress. All injections were performed on Tuesdaysand Fridays in the afternoon by a trained member ofstaff. Upon starting infliximab the mice were scoredthree times weekly (Monday, Wednesday and Friday)for inflammatory joint scores (supplementary Table 1)and collective clinical scoring of weight loss, behaviour,mobility, grimace, severity and duration of joint inflam-mation (supplementary Table 2) as previouslyreported.20

At 14 weeks old the animals were culled and theirhindlimbs were collected for histology and micro-com-puted tomography (CT) analysis.

TNFARE transgenic mouse breedingprogramme

A retrospective analysis of breeding was performed in17 breeding pairs prior to the introduction of infliximaband in 14 breeding pairs following the regular intro-duction of infliximab. Breeding pairs consisted of aC57BL/6 TNF�ARE male and a C57BL/6 wild-typefemale paired at six weeks of age. In each breedingpair, the number of successfully weaned litters duringtheir breeding lifespan, average litter size, numbersof male and female pups per litter, litters thatfailed to progress through weaning, and breedingpairs that failed to produce a litter were recorded.Breeding outputs were compared with a C57BL/6wild-type colony consisting of 10 breeding pairs, moni-tored between six and 26 weeks, at a time pointmatched to TNF�ARE animals prior to the introductionof infliximab.

Micro-CT

The hindlimbs were isolated post-mortem and imagedwith a SkyScan 1172 micro-CT scanner (Bruker,Kontich, Belgium) using X-ray beam settings of 60 kVsource voltage and 167 mA source current. Projectionswere taken every 0.45� at 600ms exposure, with animage pixel size of 13.59mm. Image volumes werereconstructed using the Feldkamp algorithm (NRecon1.6.1.5; Bruker), having applied beam hardening cor-rection. A radiodensity range of –300 to 3000 HUwas chosen to isolate the bony structures from the ima-ging medium. CTAnalyser 1.12 software (SkyScan) wasused to extract an isosurface mesh representation of thereconstructed micro-CT slices. MeshLab 1.3.2 (an opensource software developed with the support of the 3D-CoForm project) was used to modify the raw meshes,and the samples were shaded in MeshLab using ambi-ent occlusion.

Meshes were visualized in MeshLab and scoredblind by three independent researchers as follows.Meshes were divided into three regions, ‘heel’ (com-prising the calcaneus, centrale, distal tarsals, tibialeand talus but excluding the tibia and fibula), ‘metatar-sals’ and ‘phalanges’ (excluding the claws). Eachregion was scored separately for the degree of erosion:0¼ normal, 1¼ roughness, 2¼pitting, 3¼ full thick-ness holes. A score was also given to describe theextent of the area affected: 0¼ none, 1¼ a fewsmall areas, 2¼multiple small–medium sized areas,3¼multiple medium–large areas or extensive. Thetwo scores were then multiplied together for eachregion, and then summed to give a maximum scoreper paw of 27.

Histological analysis

Following decalcification in 0.5M EDTA (pH 8.0),histological analysis was performed on paraffin-embedded 10 mm sections of hindlimbs taken fromeither wild-type or TNF�ARE animals receiving inflix-imab or saline injections. The sections were stained withhaematoxylin and eosin prior to quantitative analysisof the pannus size at the metatarsal–phalangeal jointinterface using ImageJ software (NIH, Bethesda, MD,USA). Pannus size in arbitrary units (AU) wascalculated from the region of synovial pannus, clearlyvisible by histology (as depicted within supplementaryFigure 1), and invading into the subchondral bone ofthe first proximal phalanges at the metatarsal–phalan-geal joint interface. Three adjacent 10 mm sections werecut from the centre of the joint, and the pannus size wasdetermined as described above to generate a meanvalue.

Naylor et al. 61

Statistical analysis

To detect a 40% decrease in joint inflammation scoresfour experimental animals were required per group (cal-culated using in-house preliminary data in TNF�ARE

mice at week 14; score 6.6, standard deviation [SD] 1.7,power 0.8, alpha 0.05). Unless stated otherwise, datashown are mean� standard error (SE) of the mean offour wild-type control animals or four TNF�ARE ani-mals receiving saline and five animals receiving inflixi-mab. Statistical significance was defined as P< 0.05(*P< 0.05; **P< 0.005; ***P< 0.0005) using eitheran unpaired Student’s t-test or a two-way analysis ofvariance (ANOVA) with a Tukey post hoc analysiswhere a Gaussian distribution was identified or deter-mined using a non-parametric Kruskal–Wallis test witha Dunn’s multiple comparison where it was absent.

Results

Infliximab suppresses disease activityin TNFARE mice

Clinical signs of joint inflammation were evident inboth saline- and infliximab-treated mice from sixweeks of age, but were significantly reduced in thegroup receiving infliximab at eight weeks of age(Figure 1a). Joint inflammation scores remained signifi-cantly lower in the infliximab group relative to thesaline group up to 14 weeks. When combined clinicalscores measuring severity of inflammation, duration ofinflammation, mobility, behaviour, weight and grimacewere assessed, a similar pattern was observed, withincreased scores being evident in saline-treated animalsfrom eight weeks (Figure 1b). Once again, this patterncontinued up to 14 weeks.

In this study, progressive weight loss provides aneffective readout of the degree of systemic inflamma-tion. Consequently, weight loss of greater than 20%also represents an effective humane endpoint inTNF�ARE mice. TNF�ARE animals receiving salineonly, developed evidence of weight loss at day 70, plat-eauing from day 77. This was in contrast to progressiveweight gain observed throughout the infliximab-treatedTNF�ARE animals and wild-type counterparts(Figure 1c). Consequently, TNF�ARE animals receivinginfliximab maintained a normal body weight relative tocontrol animals throughout the monitoring period(Figure 1c).

Together, these data demonstrate that TNF�ARE

animals receiving regular injections of infliximab,while not completely protected from the detrimentaleffects of TNFa excess, present with a markedlymilder phenotype than untreated counterparts. This isevidenced by their significant reduction in clinicalscores of joint inflammation, improved mobility,behaviour and grimace and improved body weightsrelative to TNF�ARE animals receiving saline only.Therefore, the administration of infliximab representsa significant refinement in regard to improving animalpain and welfare in the long-term maintenance of theseanimals.

Infliximab protects against joint destruction

While scoring of inflammation and clinical features ofdisease provide valuable insight into observable meas-ures of disease activity, they are limited in their abilityto assess quantifiable measures of direct joint destruc-tion. To gain a more comprehensive understanding ofhow administration of infliximab directly influences the

Figure 1. Scoring of (a) joint inflammation, (b) clinical scoring (weight, inflammation, grimace, behaviour, mobility,inflammation severity and duration), (c) total body weights in wild-type (WT) or TNF�ARE animals receiving eithertwice-weekly intraperitoneal injections of saline or infliximab at 10mg/kg. Values are expressed as mean� standard errorof four WT animals, five TNF�ARE animals receiving infliximab and four TNF�ARE animals receiving saline. Statisticalsignificance was determined using one-way analysis of variance (ANOVA) with a Tukey’s post hoc analysis. *P< 0.05,**P< 0.005.


process of joint destruction we examined three-dimen-sional reconstructions of joints using micro-CT, andassessed joint histology of TNF�ARE animals receivingeither saline or infliximab.

Analysis of cortical bone erosion by micro-CTwithin ex vivo hind paws of TNF�ARE animals receiv-ing saline only, revealed significant full thickness holesin the talus, intermedium and centrale with markedpitting and roughness of the distal tarsals relative totheir wild-type counterparts. By contrast full thicknesscortical holes and pitting were significantly reduced inTNF�ARE animals receiving infliximab (Figure 2b).Quantification of erosions revealed a strong trendtowards reduced scores in TNF�ARE animals receivinginfliximab relative to those receiving saline which,unlike TNF�ARE animals, were not significantly greaterthan wild-type controls (Figure 2d).

The mice receiving regular infliximab injection dis-played a marked reduction in inflammatory pannus inthe metatarsal–phalangeal joint interface (Figure 2a).When the pannus invading within the subchondralbone was quantified, pannus size was found to be sig-nificantly reduced in TNF�ARE mice receiving inflixi-mab relative to those receiving saline (Figure 2c).

These data demonstrate that TNF�ARE animalsreceiving infliximab have a significant reduction injoint inflammation and destruction relative to thosereceiving saline only. Similarly, local bone erosionsand destruction of juxta articular bone withininflamed joints are also significantly reduced inTNF�ARE animals receiving infliximab. These datadirectly support clinical scoring of joint inflammationand confirm that infliximab significantly retards thedisease process that underpins deformity, loss of func-tion and joint pain.

Infliximab increases litter numbers andbreeding behaviour in TNFARE mice

To examine the impact of the introduction of infliximabon breeding behaviour and breeding output, we retro-spectively examined 17 TNF�ARE breeding pairs froma time point prior to the introduction of infliximabinjections, and 14 TNF�ARE breeding pairs followingthe introduction of infliximab injections.

The findings (Table 1) clearly demonstrated thatTNF�ARE males receiving regular infliximab injectionssired more litters over their breeding lifespan.Furthermore, when compared with wild-type controlbreeding pairs on a C57BL/6 background (Table 2),there was a significant decrease in the percentage ofTNF�ARE males successfully siring a litter. By contrast,no significant differences were identified in TNF�ARE

males successfully siring a litter following the introduc-tion of infliximab. Prior to the introduction of

infliximab injections no TNF�ARE males reached the26-week breeding lifespan due to humane endpointsbeing exceeded. By contrast, following the introductionof infliximab all except three breeding males achieved a26-week breeding lifespan (data not shown).

These data demonstrate clear benefits of regularadministration of infliximab in breeding TNF�ARE ani-mals, resulting in greater numbers of litters over theirbreeding lifespan, a greater chance of siring a litter, andtrends towards larger litter sizes and greater chances oflitters surviving past weaning. Consequently, fewerbreeding pairs are required to generate experimentalanimals, allowing a significant reduction in TNF�ARE

breeding animals needed to maintain a breedingprogramme.

Discussion

Transgenic, spontaneous mouse polyarthritis modelsare widely used within the rheumatology researchcommunity. Unfortunately, one significant drawbackin their use in medical research arises throughreduced animal welfare as a result of systemic inflam-mation in breeding animals used to generate experi-mental animals. While the 100% penetrance ofdisease symptoms in animals carrying a disease trans-gene is a benefit in helping to reduce experimentalanimal numbers, it is a detriment in breeding pro-grammes where it suppresses breeding behaviour,increases pain and distress and ultimately reducesbreeding lifespans of animals.

In this study we have explored whether the bio-logical anti-human TNFa drug infliximab may proveefficacious in improving animal welfare and breedingbehaviour in male TNF�ARE mice. Infliximab has pre-viously been shown to be highly efficacious in abrogat-ing disease activity and joint destruction in the hTNFtgmouse when given once weekly at 8mg/kg by intraper-itoneal injection, and is routinely administered in ourbreeding programmes of these animals.2,20

While hTNFtg and TNF�ARE models of polyar-thritis are driven by a common pathophysiology,infliximab is not reported to cross react with murineTNFa.21 However, its efficacy has been reported inseveral murine models of chronic inflammation sug-gesting further modes of action beyond direct neutral-ization and depletion of TNFa.15–19 These include theinduction of T-cell apoptosis and suppression ofangiogenesis, possibly mediated via Fc-mediated inter-actions and the natural immunogenicity ofinfliximab.21–24

Therefore, we predicted that infliximab may proveeffective in suppressing disease activity in TNF�ARE

animals in an identical manner as reported in hTNFtgmice.25 Previous studies using infliximab in mice have

Naylor et al. 63

demonstrated that it is well tolerated at levels of upto 10mg/kg over a period of up to 24 weeks.26

Consequently, following consultation with the namedanimal care and welfare officer and named veterinaryofficer within our animal handling facility, we examinedwhether twice-weekly 10mg/kg intraperitoneal

injections of infliximab would improve animal welfareand breeding in TNF�ARE animals.

We found that clinical scoring of joint inflammation,duration of inflammation, mobility, behaviour andbody weight were all significantly improved inTNF�ARE animals receiving infliximab relative to

Figure 2. (a) Representative paraffin-embedded sections from the metatarsal–phalangeal joint interface stained withhaematoxylin and eosin (scale bars 200 mm). (b) Representative images of 3D reconstructions of hind paws with increasedmagnification at the ankle, using micro-computed tomography(CT). (c) Quantification of degree of pannus invasion in themetatarsal–phalangeal joint interface (from haematoxylin and eosin histology). (d) Quantification of cortical erosion in thebones of the ankle, metatarsals and phalanges from micro-CT in wild-type (WT) or TNF�ARE animals receiving eithertwice-weekly intraperitoneal injections of saline or infliximab at 10mg/kg. Values are expressed as mean� standard errorof four WT animals, five TNF�ARE animals receiving infliximab and four TNF�ARE animals receiving saline. Statisticalsignificance was determined using one-way analysis of variance (ANOVA) with a Tukey’s post hoc analysis for pannusinvasion and Kruskal–Wallis test and Dunn’s multiple comparison for micro-CT quantification of erosions. Arrows denotepannus adjacent to cortical bone in (a), and full thickness cortical bone erosions within (b). **P< 0.005, NS: not significant.


those receiving saline alone. Unfortunately, as opposedto administration of infliximab in hTNFtg animals,clinical signs of polyarthritis were still evident, indicat-ing that regular infliximab injections were unable tocompletely abrogate host murine TNFa overexpres-sion. Infliximab was originally designed to target anddeplete human TNFa, and therefore may utilize alter-native modes of action in suppressing disease activity ininflammatory models driven by murine TNFa.Therefore, it is unsurprising that infliximab injectionsare less effective at suppressing disease in this model.Despite this, it is clear that regular administration ofinfliximab significantly suppresses polyarthritis diseaseactivity and improves animal welfare. This was moreevident when the structure of bone by micro-CT and

the histology of joints were examined, showing signifi-cantly reduced pannus invasion and bone erosions inaffected joints.

While disease activity was not completely suppressedin TNF�ARE animals receiving infliximab, bodyweights were markedly increased relative both tothose receiving saline only and to their wild-type coun-terparts. Weight loss in TNF�ARE animals can in partbe attributed to malnutrition secondary to intestinalinflammation. Whether the weight gain observed inthe infliximab-treated group reflects the suppressionof inflammatory bowel disease-like symptoms cannotbe determined from this study without examining intes-tinal pathology. This is further complicated, as rapidweight loss is also an important measure of

Table 1. Mean (AVG) number of litters successfully weaned, mean number of pups generated per litter, mean number ofmale and female pups per litter, percentage of litters that successfully survive beyond weaning and percentage of malesthat successfully sire a litter in breeding pairs consisting of heterozygous TNF�ARE males and wild-type (WT) females overperiods preceding the introduction of regular infliximab injections, or following the regular introduction of twice weeklyintraperitoneal injections of infliximab at 10mg/kg in breeding males.

TNF�ARE male�WT female

Pre infliximab Post infliximab

AVG SE AVG SE Significance

Litters weaned 1.69 0.22 3.00 0.19 0.0019

Total pups per litter 5.66 0.47 6.69 0.48 0.085

Males per litter 2.61 0.32 3.45 0.39 0.086

Female per litter 3.05 0.38 3.24 0.29 0.635

Litters successfully weaned (%) 74.13 8.65 93.46 1.28 0.0604

Successfully sire a litter (%) 70.60 14.87 85.72 9.70 0.282

Values are expressed as mean� standard error (SE) from 17 breeding pairs where male TNF�ARE animals did not receive infliximabinjections versus 14 breeding pairs where male TNF�ARE animals received regular infliximab injections. Statistical significance wasdetermined using an unpaired student’s t-test for litter size and pups for litter and a Mann–Whitney U-test for percentage of littersweaned and percentage successfully siring a litter.

Table 2. Mean (AVG) number of litters successfully weaned, mean number of pups generated per litter, mean number ofmales and female pups per litter, percentage of litters that successfully survive beyond weaning and percentage of malesthat successfully sire a litter in breeding pairs consisting of wild-type (WT) males and WT females over the periodspreceding the introduction of regular infliximab injections.

WT male�WT female

WT breedingVersus TNF�ARE

Versus TNF�ARE/infliximab

AVG SE Significance Significance

Litters weaned 3.80 0.39 0.0016 0.065

Total pups per litter 6.43 0.80 0.27 0.63

Males per litter 3.41 0.53 0.10 0.93

Female per litter 3.02 0.39 0.95 0.54

Litters successfully weaned (%) 86.77 10.16 0.883 0.178

Successfully sire a litter (%) 100 0.00 0.136 0.146

Values are expressed as mean� standard error (SE) from 10 breeding pairs. Statistical significance was determined using an unpairedstudent’s t-test for litter size and pups for litter and a Mann–Whitney U-test for percentage of litters weaned and percentage successfullysiring a litter.

Naylor et al. 65

deteriorating health in cytokine-driven cachexia. TNFadirectly induces numerous catabolic pathways in miceand humans, and also suppresses normal feedingbehaviour.27–29 Regardless of the underlying mechan-ism, when combined with our observations with diseaseactivity and joint destruction, this study clearly demon-strates that regular administration of infliximab inTNF�ARE animals represents a significant refinementin their maintenance in breeding programmes, andmarkedly improves animal welfare.

Retrospective examination of the impact of admin-istration of infliximab in TNF�ARE breeding malesdemonstrated that it was highly efficacious in improv-ing breeding behaviour, increasing the number of litterssired during their breeding lifespan and increasing thepercentage of males that successfully bred and siredlitters. In addition, non-significant trends wereobserved in the numbers of pups sired per litter, favour-ing increased numbers of males per litter. This resultwas perhaps unsurprising given that these mice survivelonger without exceeding the criteria for their humaneendpoints; and it also undoubtedly reflects an improve-ment in animal welfare through reduced pain and dis-tress – factors that are known to suppress matingbehaviour in male rodents.10

Taken together, these results clearly demonstratethat the application of regular infliximab injections inTNF�ARE breeding males represents a significantrefinement in practice, allowing a reduction in animalnumbers required in routine breeding to generateexperimental animals.

A final consideration should be whether regularinfliximab administration would prove beneficial inother genetically manipulated models of spontaneouspolyarthritis, such as the KBxN, SKG and DNase IImice. These models have varying involvement forTNFa in their disease pathology, and so the beneficialeffects may vary greatly. Consequently, we proposethat its application in these models might meritinvestigation.

Conclusions

This study is the first to report that regular administra-tion of infliximab is effective at suppressing diseaseactivity systemic inflammation and improving animalwelfare in TNF�ARE animals. In addition, we haveshown that infliximab is highly efficacious in improvingbreeding behaviour and increasing the number of litterssired by TNF�ARE males. Consequently, in bothhTNFtg and TNF�ARE mice, regular administrationof infliximab in breeding males represents a refinementin routine maintenance and allows a significant reduc-tion of the numbers of breeding animals required togenerate experimental animals.

Acknowledgements

We would like to thank Professor George Kollias (Hellenic

Pasteur Institute, Athens, Greece) for providing the hTNFtgand TNF�ARE mice; the Biomedical Services Unit (Universityof Birmingham) for supporting animal experiments; and theDepartment of Musculoskeletal Pathology (Robert Aitken

Institute, University of Birmingham) for embedding and cut-ting tissue for histology.



Funding

The author(s) disclosed receipt of the following financial sup-

port for the research, authorship, and/or publication of thisarticle: This research was supported by Arthritis ResearchUK grants (Reference: 19859 & 20843).

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Resume

Des modeles transgeniques TNFa de polyarthrite tels que la souris TNF�ARE se sont reveles precieux pourdelimiter les aspects de la pathophysiologie des maladies inflammatoires chez les humains.Malheureusement, l’apparition de l’inflammation et la destruction des articulations chez ces modeles com-promet gravement la gestion de la reproduction. Nous avons cherche a savoir si un traitement depletif TNF apar « Infliximab » pourrait constituer une nette amelioration pour la reproduction de routine. Les resultatscliniques de l’inflammation des articulations ont ete evalues chez les males TNF�ARE recevant soit del’Infliximab (10mg/kg) soit une solution saline par injection intraperitoneale deux fois par semaine.L’histologie des articulations et la morphologie des os ont ete respectivement evaluees par une analysehistologique et par micro-scanner. L’analyse de la reproduction a ete realisee retrospectivement chez lesmales TNF�ARE avant et apres l’administration reguliere d’infliximab. Les resultats cliniques d’inflammationont ete reduits significativement chez les males TNFDRE recevant de l’Infliximab (controle 6.6 AU þ 0.88contre infliximab, 4.4 AU� 1.4; p< 0.05), tandis que les mesures d’invasion du pannus et de l’erosion desos par histologie et micro-scanner ont ete nettement reduites. Chez les groupes reproducteurs, les malesTNF�ARE recevant des injections d’infliximab ont engendres plus de portees pendant leur duree de viereproductive (controle 1.69� 0.22 contre infliximab, 3.00� 0.19; p< 0.005). Avant l’infliximab, les males

Naylor et al. 67

TNF�ARE avaient en outre 26% de risques de ne pas pouvoir se reproduire. Ce risque a ete reduit de 7% apresl’administration d’Infliximab. Cette etude est la premiere a rapporter que l’administration reguliered’Infliximab est efficace pour supprimer l’activite de la maladie et pour ameliorer le bien-etre des animauxTNF�ARE. De plus, nous avons montre que l’infliximab est tres efficace pour ameliorer le comportementreproductif et pour augmenter le nombre de portees engendrees par les males TNF�ARE.

Abstract

Transgene TNFa-gestutzte Polyarthritismodelle wie die transgene TNF�ARE Maus haben sich zur Darstellungder Pathophysiologie entzundlicher Krankheiten des Menschen als außerst wertvoll erwiesen. Leider stelltdas Auftreten von Gelenkzerstorung und -entzundung bei diesen Modellen eine erhebliche Beeintrachtigungdes Zuchtmanagements dar. Wir untersuchten, ob TNFa-Blocker-Therapie eine wesentliche Verbesserungroutinemaßiger Zucht bewirken kann. Klinische Gelenkentzundungswerte wurden bei TNF�ARE Mannchenuntersucht, denen entweder Infliximab (10mg/kg) oder Salzlosung mittels zwei Mal wochentlich erfolgenderintraperitonealer Injektion verabreicht wurde. Gelenkhistologie und Knochenmorphologie wurden jeweilsdurch histologische Analyse und Mikro-CT bewertet. Die Zuchtanalyse wurde retrospektiv bei TNF�ARE

Mannchen vor und nach dem regelmaßigen Einsatz von Infliximab untersucht. Klinische Entzundungswertewaren bei TNF�ARE Mannchen, die Infliximab erhielten, signifikant reduziert (Kontrolle 6.6 AU� 0.88 versusInfliximab, 4.4 AU� 1.4; p< 0.05), wahrend mittels Histologie und Mikro-CT gewonnene Messwerte vonPannusbildung und Knochenerosion deutlich reduziert waren. In den Zuchtgruppen zeugten TNF�ARE

Mannchen, die Infliximab erhielten, mehr Wurfe wahrend ihrer Zuchtdauer (Kontrolle 1.69� 0.22 versusInfliximab, 3.00� 0.19; p< 0.005). Zudem bestand bei TNF�ARE Mannchen vor Infliximab-Erhalt ein 26%-iges Risiko, keinen Nachwuchs zu zeugen. Nach Infliximab-Einsatz sank dieser Wert auf 7%. Mit dieserStudie wird erstmals berichtet, dass regelmaßige Infliximab-Gabe eine Hemmung der Krankheitsaktivitatund eine Verbesserung des Wohlbefindens von TNF�ARE Tieren bewirkt. Außerdem wurde gezeigt, dassInfliximab hochst wirksam zur Verbesserung des Zuchtverhaltens und zur Erhohung der Zahl der vonTNF�ARE Mannchen gezeugten Wurfe beitragt.

Resumen

Los modelos transgenicos de poliartritis impulsados por TNFa como el raton TNF�ARE han demostrado sermuy valiosos para delinear aspectos de la patofisiologıa de enfermedades inflamatorias en humanos.Lamentablemente, el principio de destruccion de articulaciones e inflamacion en estos modelos representaun detrimento significativo para la gestion de crıa. Examinamos si la terapia de agotamiento de TNFa‘‘Infliximab’’ puede representar un refinamiento significativo en la crıa rutinaria. Se evaluaron distintaspuntuaciones clınicas de inflamacion de articulaciones en machos TNF�ARE que recibıan Infliximab (10mg/kg) o salina dos veces a la semana mediante inyeccion intraperitoneal. La histologıa de las articulaciones y lamorfologıa osea fueron evaluadas mediante analisis histologicos y micro-CT respectivamente. Se examinaronanalisis de la crıa retrospectivamente en machos TNF�ARE antes y despues de la introduccion regular deInfliximab. Las puntuaciones clınicas de inflamacion se redujeron significativamente en los machos TNF�ARE

que recibıan Infliximab (control 6.6 AU� 0.88 versus Infliximab, 4.4 AU� 1.4; p< 0.05), mientras que lasmedidas de invasion de pannus y erosion osea mediante histologıa y micro-CT se redujeron significativa-mente. En los grupos de crıa, los machos TNF�ARE que recibıan inyecciones de Infliximab crearon mascamadas en su vida util de crianza (control 1.69� 0.22 versus Infliximab, 3.00� 0.19; p< 0.005). Asimismo,antes de Infliximab, los machos TNF�ARE tuvieron un 26% de riesgo de no poder crear ninguna camada. Estose redujo en un 7% tras la introduccion de Infliximab. Este es el primer estudio en demostrar que la admin-istracion regular de Infliximab resulta eficaz para suprimir la actividad de enfermedades y para mejorar elbienestar de animales TNF�ARE. Asimismo, hemos demostrado que Infliximab es altamente eficaz paramejorar el comportamiento reproductor y para aumentar el numero de camadas creadas por machosTNF�ARE.


ab oratory

l i m i t e d

lan imals

Original Article

Laboratory animal science coursein Switzerland: participants’ pointsof view and implications for organizers

Fabienne Crettaz von Roten

AbstractSwitzerland has implemented a mandatory training in laboratory animal science since 1999; however a com-prehensive assessment of its effects has never been undertaken so far. The results from the analysis ofparticipants in the Swiss Federation of European Laboratory Animal Science Associations (FELASA) CategoryB compulsory courses in laboratory animal science run in 2010, 2012, 2014 and 2016 showed that the par-ticipants fully appreciated all elements of the course. The use of live animals during the course was supportedand explained by six arguments characterized with cognitive, emotional and forward-looking factors. A largemajority considered that the 3R (replacement, reduction and refinement) principles were adequately appliedduring the course. Responses to an open question offered some ideas for improvements. This overall positivepicture, however, revealed divergent answers from different subpopulations in our sample (for example,scientists with more hindsight, scientists trained in biology, or participants from Asian countries).

Keywordseducation and training, course evaluation, live animals, 3Rs, scientists’ survey

Date received: 26 January 2017; accepted: 17 March 2017

Switzerland is a leading country in the pharmaceuticaland biotechnological sector, with a network of top-quality academic institutions focused on basic researchand education in the life and biomedical sciences.Switzerland still relies on animal experimentation forits basic and translational research. In 2015, 682,333experimental animals were used; the number of experi-mental animals used dropped substantially in the 1990s(from 1,177,778 in 1990 to 588,778 in 1999), but it roseslightly in the early 2000s (to 681,410 in 2004) and hassince stayed at around 600,000 animals.1

Switzerland has stringent regulations regardinganimal protection,2 which significantly exceed theEU’s standards. Since 1999, a mandatory laboratoryanimal science (LAS) course has been introduced forevery scientist seeking to perform animal experimenta-tion in Switzerland. This course was later accredited,first in the German-speaking region of Switzerland in2008 by the Federation of European LaboratoryAnimal Science Associations (FELASA) (and re-accre-dited under the EU ‘Functions’ scheme in 2015; currentFELASA identification: F027/06), and then in theFrench-speaking region in 2013 (current FELASA

identification: 038/12) (see http://www.felasa.eu/accred-itation-boards/accreditation-board-for-education-and-training1/felasa-accredited-courses2/ [accessed 2 April2017]). Both courses have been aimed at people whoneed to apply procedures to animals, described as EUFunction A for the former and FELASA Category Bfor the latter. (See Appendix 1: The FELASA‘Categories’ and Directive ‘Functions’, a historicalreminder of the paper to be downloaded under thesection ‘I am a course provider. How do I get mycourse accredited?’ at http://www.felasa.eu/accreditation-boards/accreditation-board-for-education-and-training1/ [accessed April 2, 2017].) At the Europeanlevel, the European Commission’s Directive 2010/63/EU requires, among other things, that researchersundertake specific training in order to be licenced to

Observatoire Science, Politique et Societe, Universite de Lausanne,Lausanne, Switzerland

Corresponding author:Fabienne Crettaz von Roten, Universite de Lausanne, SSPBatiment Geopolis Lausanne, CH-1015 Lausanne, Switzerland.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 69–78



sagepub.co.uk/


DOI: 10.1177/0023677217708807


practise animal experimentation.3 The EuropeanCommission has issued a series of guidance documentsto help its Member States to translate the Directive intoeffective measures for the protection of animals used forscientific purposes.4,5 Among these, the Commissionhas published a guidance document for the develop-ment of a common education and training frameworkwhich was heavily inspired by FELASA’s recommenda-tions for the courses of Category B or C researchers.4

It is legally forbidden in Switzerland to use animalswith constraints in education when there are other waysof teaching the skills needed to conduct an in vivo study(Art. 138, let. 1 al. c6). Another piece of legislation (Art.3, let. C, al. 67) considers the use of live animals ineducation and training to be an experiment.Therefore, the number of animals, the procedures, thereasons why live animals are needed for skill training,and a cost–benefit assessment have to be provided bythe course organizers to the ethical review board forevaluation and approval by the local competent autho-rities. The applicant will also need to show how the 3Rprinciples – replacement, reduction and refinement –will be implemented in the course.

Swiss legislation requires that skill training on liveanimals should be delivered during the course. Thetraining should include handling, restraint, weighing,sexing, identification methods of animals, blood, urineand faeces sampling, as well as injections of substances.It is also required that basic procedures in anaesthesiaand analgesia are covered in the training (Art. 25, RS455.109.18).

The course in Switzerland uses a tiered approach inthe use of live animals by starting with non-animalalternatives, such as videos, progressing to cadaversand finally to live animals. However, the number ofanimals used in education and training is generallyincreasing in Switzerland (4095 mice and rodents in2000, 5317 in 2010, 5131 in 2014 and 5309 in 2015).1

This is due to the increasing numbers of students andscientists who need to attend this course, and becausecourse organizers do not want to impose multipleprocedures on any single animal, which highlights theclassical dilemma between reduction and refinement.9

The fact to use live animals in education and trainingprogrammes is a long-standing issue. As a sequel to theEU Directive, the European Commission has releasedsome guidance on this issue in its recent publication onproject evaluation and retrospective assessment.5

After almost 30 years of the mandatory implementa-tion of LAS education and training in Switzerland andthe fact that a considerable number of people havereceived this training, we felt it was important to assessparticipants’ attitudes towards the course, particularlywith regard to the 3Rs and the use of live animals ineducation and training. More specifically, we were

interested in the participants’ backgrounds and theirsubsequent careers after the course. What were theirattitudes towards the use of live animals in the courseafter a few years of professionally practising animalexperimentation? What did the participants wish toimprove or change about the course retrospectively?

Little is known about the scientists’ points of viewon these issues. There have been some quantitative stu-dies on scientists’ attitudes towards the 3Rs,10–13 andon general attitudes towards the LAS course.14 On atti-tudes towards the use of animals during the LAScourse, there have been a quantitative study on under-graduate and postgraduate students15 and a qualitativestudy,16 both in Sweden. However, there have been nostudies focusing on all types of participants, or specifi-cally on Switzerland. In 2016 a study was designed toanswer these research questions, and a selection of itsresults is presented here.


This study was designed in partnership with the Swissnetwork on Education in Laboratory Animal Science,i.e. its French section, the ‘Reseau des Animaleries del’Arc Lemanique’ (ResAL) and its German section,the ‘Institut fur Labortierkunde Universitat Zurichand ETH Zurich’ (LTK), both of which providedinformation on the participants of the course. Thetarget population was the participants of theFELASA-accredited Category B course on rodents(mice and rats) held in Switzerland, because theseanimals corresponded to around 75% of the totalnumber of animals used. The course includes 20 hof lectures and 20 h of practical training with topicsdefined by the Swiss legislation, and accredited as theFELASA B course.

This study aimed to record the attitudes of theparticipants with various levels of hindsight towardsthe course, i.e. from participants who had recently fin-ished the course to those who had been practising whatthey had learned during the course for a few years.Therefore, we opted for a stratified sample. To ensurea valid assessment of the course and to avoid memorybias, it was decided not to go too far back in time andto opt for four strata defined by the calendar year of thecourse (cohorts 2010, 2012, 2014, and 2016). For eachyear, a sample of courses were selected.

An online survey was sent to the participants of theselected courses, using the email addresses provided bythe training officials. An invitation was sent to the par-ticipants in January 2016 for the 2016 cohort, andbetween June and July 2016 for the three other cohorts.In total, 1451 emails were sent, but 387 were returneddue to email addresses being invalid (mostly in the 2010and 2012 cohorts), illustrating the high mobility of


scientists. A total of 510 respondents completed theonline survey, which corresponded to a 48% responserate.

The questionnaire featured 28 questions organized inthree sections: the first focused on the demographics ofthe respondent, the second on the course – particularlyon the use of live animals, and the third on the 3Rs.

The sociodemographic variables were gender; age(recoded into four age groups: 19 to 25, 26 to 31, 32to 36, and above 37 years old); and nationality (recodedinto five groups of countries: Switzerland, Europeancountries, Asian countries, North American countries,and the rest of the world).

The academic variables were the linguistic regionswhere the course was taken (German- or French-speaking); the disciplines of the participants’ firstdegrees (recoded into five groups: biology; chemistry,biochemistry, physics, biophysics or pharmaceuticalsciences; medicine; veterinary medicine; and other); adichotomous variable for their PhD status (‘Yes’ or‘No’); the participants’ occupations at the time of thesurvey (six possibilities: lab technician; bachelor’s ormaster’s student; PhD student; postdoc or seniorscientist; principal investigator, faculty teacher andphysician; and other); the participants’ internationalexperience (a series of ‘Yes’ or ‘No’ questions on fourpossibilities of international experience: during theirbachelor’s or master’s studies, during their PhD,during their postdoc experience, or during theiremployment or academic position with a minimum ofone year. This last set of items was then recoded into adichotomous variable indicating whether the respon-dent had one or more international experience or none).

In the second section, three five-point Likert-typeitems were used for the assessment of the course(from ‘very good’ to ‘very poor’), and for attitudestowards the use of animals and the 3Rs (from ‘stronglydisagree’ to ‘strongly agree’). An aggregate scale wasbuilt on the three items of assessment, with higherscores indicating a more positive assessment.

For the open question on animal use, two indepen-dent researchers analysed the responses, and found sixdimensions which were consistent between the tworesearchers. The open question’s answers were thenrecoded in accordance with these six dimensions into amultiple-answer variable because some respondents hadprovided an answer with multiple arguments. Therefore,the sum of the percentages for each type of argument ishigher than 100%.

Descriptive statistics were performed on eachvariable. Chi-square independence tests were per-formed to assess the differences between cohorts,gender, linguistic regions, nationalities, disciplines andoccupation. When the conventional rule of thumb ofless than 25% of cells with expected numbers less than

five was not verified, a recoding was undertaken. Forexample, there were too few veterinarians to be ana-lysed separately therefore we considered respondentswith first degrees in medicine and in veterinary medi-cine together for some analysis. We also recoded thesmallest nationality groups, i.e. North American coun-tries and the rest of the world, together. If recoding wasnot possible, we used resampling techniques (MonteCarlo methods). Because not all respondents answeredall of the questions, the group sizes may vary betweenquestions. P values of less than 0.05 were considered tobe statistically significant. Two-way analysis of var-iance (ANOVA) were used to analyse the effects oflinguistic regions and nationalities, disciplines andnationalities on the aggregated scale of assessment ofthe course. We used a heteroscedasticity-corrected coef-ficient from the library ‘car’ of the R software if theLevene test of homogeneity of variance was rejected.Other statistical analyses were performed using SPSS23 software (IBM Corp, Armonk, NY, USA).

Results

Sample characterization

Among the respondents to this study, 79% were fromthe German-speaking region and 62% were female(Table 1). The ages ranged between 19 and 62 yearsold, with a mean age of 32 years old (standard deviation[SD] 6.1). The mean age relative to the cohort’s yeardecreased slightly (from 30.8 years in 2010 to 29.4years in 2016). Among the respondents, 33% wereSwiss and 51% were of other European nationalities.

Professionally, 68% of the respondents indicatedhaving a first degree in biology, whereas 15% indicatedhaving one in chemistry or biochemistry or physics orpharmaceutical sciences, and 13% in medicine. On thewhole, 40% of respondents had a PhD. Among thoseemployed, 36% were PhD students. Furthermore, 34%were postdocs or scientists with a PhD; 12% were labtechnicians; and 9% were faculty teachers, principalinvestigators or physicians. Finally, with regard to thecountry where they lived when they were 18 years old,19% of the respondents studied in a different country,31% earned their PhD in a different country, 32% hadpostdoc experience in a different country, and 36% hada job in a different country. On the whole, 56% of therespondents had at least one international study orwork experience.

Among those who had performed animal experi-ments so far, a majority of respondents indicated theyonly had ethical doubts or concerns occasionallyregarding animal use in their work (62%), whereas10% frequently had doubts or concerns, and 27%had no doubts or concerns.

Crettaz von Roten 71

Views on the course

The general assessment of the course presented a posi-tive picture. A large majority of respondents were infavour of this mandatory course (93%, among whom82% were strongly in favour, Table 2). The course waspositively rated (91%, Table 2), and with regard to the

balance between theory and practical work (89%), andthe practical training on animals (87%).

These positive views were very consistent among thesubpopulations, bearing no relationship to gender, agegroup, occupation or level of internationalization. Onesignificant difference was found among the linguisticregions (P< 0.01): those taking the course in theGerman-speaking part of the country were even morepositive about the course than their French counter-parts (93% of the German-speaking region indicated‘good and very good’ against 84% from the French-speaking region, Table 2). There was also a significantdifference among the disciplines (P< 0.05): thosehaving studied their first degrees in veterinary medicineor medicine were the most positive about the practicaltraining (96% indicating ‘good and very good’, com-pared with only 86% of those with biology degrees,who were the least positive, Table 2). The geographicalorigin of participants revealed three significant differ-ences for people from Asia compared with those fromthe three other regions: those in Asia were less positivetowards the mandatory course (P< 0.001, 78% ofagreement against 91% to 97% in the three othergroups), towards the course in general (P< 0.05, 86%of ‘good and very good’ against 91% to 92%), andtowards the balance between theory and practicalwork (P< 0.05, 82% against 85% to 92%).

There was a significant difference of repartitionamong nationalities in the two linguistic regions(�2(4)¼ 17.94, P< 0.001: higher percentages ofEuropean and Asian respondents in the French-speak-ing region, and a higher percentage of Swiss in theGerman-speaking region). The aggregated scale ofassessment varied between 4 and 15 (M¼ 12.96,SD¼ 1.77). Linguistic regions still had an effect onthis scale, after controlling for nationality on thisscale (F(1,438)¼ 9.33 P< 0.01). There was also a sig-nificant main effect of nationality (F(3,407)¼ 25.65,P< 0.001: the minimum average was for the respon-dents with an Asian nationality), and of disciplines(F(2,407)¼ 4.28, P< 0.05: the minimum average wasfor the respondents having studied biology), and a sig-nificant interaction between nationality and discipline(F(6,407)¼ 3.11, P< 0.01: the minimum average wasfor biology for all nationalities, except for the Swisswhere this was for medicine and veterinary medicine).

Views on the use of live animals duringthe course

Secondly, scientists were asked about the use of liveanimals, and their responses indicated that theyapproved this approach (Figure 1). A large majorityagreed that the course increased their awareness ofanimal welfare (87% answered ‘strongly and tend to

Table 1. Demographics of respondents.

Respondent group n¼ 510

Sex (Female) 314 (62%)

Age

19–25 years 34 (7%)

26–31 years 257 (50%)

32–36 years 126 (25%)

> 36 years 93 (18%)

Nationality

Swiss 166 (33%)

European countries 260 (51%)

Asian countries 51 (10%)

North American countries 15 (3%)

Rest of the world* 17 (3%)

Linguistic region (German-speaking) 403 (79%)

Discipline of first degree

Biology 330 (68%)

Chemistry, biochemistry, physics, bio-physics, pharmaceutical sciences

72 (15%)

Medicine 63 (13%)

Veterinary medicine 17 (4%)

Othery

Main occupation

Lab technician 59 (12%)

Bachelor’s/Master’s student 34 (7%)

PhD student 184 (36%)

Postdoc, senior scientist 170 (34%)

Faculty teacher, principal investigator,surgeon, physician

44 (9%)

Other§ 19 (3%)

International experience 235 (56%)

During the studies 90 (19%)

During PhD 142 (31%)

During postdoc 141 (32%)

During employment 163 (36%)

*For some analyses, the groups ‘North American countries’ and‘Rest of the world’ have been gathered together (6%). yFor someanalyses, the group ‘Other’ has been excluded, and the groups‘Medicine’ and ‘Veterinary medicine’ have been gathered together(16%). §Among the 19 scientists who ticked ‘Other’, five (1%) indi-cated that they were unemployed. For some analyses, ‘Other’ hasbeen excluded, the groups ‘Lab technician’ and ‘Bachelor’s/Master’s student’ have been gathered together (19%), the groups‘Postdoc, et al.’ and ‘Faculty teacher, et al.’ have been gatheredtogether (44%).


agree’) and their personal skills (88%), and disagreedthat the course raised discomfort, fears or phobias(75%) or raised concerns about the death of theanimal at the end (56%). On the question of ethicalproblems, the opinions were divided (36% disagreed,35% agreed and 29% were ambivalent).

A course without the use of live animals?

A large majority of respondents (75%) rejected thesuggestion that a course without live animals wouldhave still enabled them to perform their experimentsafterwards to the same level of quality. This opinionwas not related to gender, discipline, age group, occu-pation, nationality or level of internationalization. Onedifference was found between the linguistic regions(P< 0.05): those who had taken the course in theGerman-speaking part of the country disagreed morefrequently than those in the French-speaking region(78% against 66%).

Among the respondents who favoured the use of liveanimals in the course, 73% explained their choice in an

open question. A content analysis of their responsesdelineated six dimensions, each of which was anargument in favour of the use of live animals inthe course. The first argument was the most impor-tant in the whole sample (around 40%), and in eachsubgroups defined by sociodemographic and aca-demic factors. However, some ranking differenceswere observed for the second and third most impor-tant arguments among the subgroups. Listed beloware each of the arguments in their order of impor-tance: the first two and the fourth arguments couldbe characterized as cognitive factors, the fifth as anemotional factor, and the third and sixth as forward-looking factors.

1. The importance of practice (versus theory) to increaseskills (cognitive factor, 69% of total responses)

One can only really learn through practising with,handling, and manipulating live animals, and practicesignificantly increased skills. These respondents draw astrong distinction and hierarchy between the learning

Table 2. General assessment of the course.

Stronglyoppose

Tend tooppose Neither

Tend tofavour

Stronglyfavour

Decision of a mandatory course inlaboratory animal (nc¼ 507)

1.6% 1.6% 3.4% 11.6% 81.9%

Swiss 1.8% 1.2% 3% 9.6% 84.3%

European countries 1.6% 0% 1.9% 11.6% 84.9%

Asian countries 0% 12.0% 10% 20% 58%

North America and rest of the world 3.1% 0% 6.3% 9.4% 81.3%

Very poor Poor Average Good Very good

The course (nc¼ 504) 0.4% 1% 7.3% 41.9% 49.4%

German-speaking 0.5% 0.8% 5.5% 39.5% 53.8%

French-speaking 0% 1.9% 14.4% 51% 32.7%.......................................................................................................................................................................................................Swiss 0% 0.6% 7.3% 42.1% 50%

European countries 0.4% 0% 7.4% 43.6% 48.6%

Asian countries 0% 4% 10% 44% 42%

North America and rest of the world 3.1% 3.1% 3.1% 25% 65.6%

The balance between theory andpractical work (nc¼ 506)

0.6% 1% 8.7% 44.7% 45.1%

Swiss 0% 0% 7.8% 46.4% 45.8%

European countries 0.4 0.8% 8.1% 46.5% 44.2%

Asian countries 0% 4.1% 14.3% 38.8% 42.9%

North America and rest of the world 6.3% 0% 9.4% 31.3% 53.1%

The practical training on animals (nc¼ 450) 0.2% 1.1% 11.3% 46.7% 40.7%

Biology 0.3% 0.7% 12.7% 47.1% 39.2%

Chemistry, et al. 0% 4.6% 6.2% 46.2% 43.1%

Medicine and veterinary medicine 0% 0% 4.3% 46.4% 49.3%


opportunities related to ‘theory’ and to ‘practice’. Thisargument was expressed the most by those with a firstdegree in veterinary medicine (among the responses ofvets, 82% chose this argument) but was expressed theleast by people with a first degree in medicine (64%);the most by lab technicians (88%) but the least bybachelor’s or master’s students (61%); the most in theGerman-speaking region (71%) and the least in theFrench-speaking region (60%).

Here is a selection of answers belonging to thisdimension:

. ‘Reaction and behaviours of animals can never betheoretically explained. Hands-on work isnecessary.’

. ‘This applies to whatever experience in life. Theory isvery important to develop awareness and criticalsense, but one definitely learns by practice!’

2. The role of tutors during the first manipulation oflive animals (cognitive factor, 17% of totalresponses)

The second most frequent argument focused onthe guidance of the tutors as opposed to the studydirectors in terms of helping the participants acquirethe proper gestures and manipulations in accordancewith the latest regulations. Among this group ofresponses, some outlined the lack of availability andof up-to-date knowledge from the study directors ortheir deputies in their labs. This argument wasexpressed the most by women (25% of womenagainst 18% of men), by respondents from theGerman-speaking region (24% of the German-speak-ing against 15% of the French-speaking), by bache-lor’s and master’s students (44%) and respondents

with a North American nationality (56%), and wasexpressed the least by lab technicians (8%) and par-ticipants of Asian nationalities (4%).

. ‘Doing the course with animals has the strongadvantage of being supervised by experiencedpeople who will tell me what I do wrong and likethat I will improve my techniques.’

. ‘It is important to learn from experts how to handlelive animals and how to perform the work in accor-dance with the most up-to-date regulations (injec-tions, proper anaesthesia, etc.). Learning directlyfrom the supervisor (i.e. study director) in the lableads to incomplete formation, lack of awarenessof newer regulation, variable standards betweenlabs, etc.’

3. Using live animals for the benefits of animals used infuture research (forward-looking factor, 13% of totalresponses)

The third most frequent argument highlighted thebenefits for animals – learning better handling, whichcauses less harm and stress to animals in futureresearch, and in fine which uses fewer animals. Thisargument was the second most cited for respondentsoccupying a position as a principal investigator, facultyteacher or physician (25%) or lab technician (13%); forthose in the French-speaking region (17%); for respon-dents with first degrees in medicine (19%); and in chem-istry, biochemistry, physics, biophysics orpharmaceutical sciences (21%).

. ‘I got more confident about handling animals duringthe course, what is for sure beneficial for the animalsI am handling now.’

0% 50% 100%

The course made me more aware of the issueof animal welfare. (nc = 508)

The use of live animals facilitates significantprogress in my skills. (nc = 508)

This course raised some discomfort, fears,phobia by using live animals. (nc = 508)

I was uncomfortable to know that the animals Itrain on would be killed in the end. (nc = 507)

This course raised some ethical problems byusing live animals. (nc = 508)

StronglydisagreeTend todisagreeNeither

Tend to agree

Stronglyagree

Figure 1. Attitudes towards the use of live animals.


. ‘The learning course would have been less steep, somore experimental animals would have suffered untilI could perform all procedures.’

. ‘In the long term it is better to be trained once thanusing more animals due to the lack of training.’

Finally, we found the last three arguments, whicheach recorded around 9% of responses (see 4–6 below).

4. The importance of reality—as opposed to simulation,video, etc.—in practice (cognitive factor, 9% of totalresponses)

The use of live animals put participants in a ‘realsituation’ and gave them ‘real and live experience’very similar to their future work in labs, allowing thedevelopment of practical knowledge. This ‘reality’ isinvaluable and cannot be replicated by other learningmodalities (simulations, puppets, dead animals and soon): when the latter modalities are used, the skills arenot fully mastered, and the participant has to relearnthem.

. ‘Hands-on training is remembered differently thanjust watching a video or seeing a picture.’

. ‘The quickness to fix a mouse or the weight of rator thickness of the skin or moves and reactionswhen administering an injection cannot be studiedtheoretically, on a puppet or even an already deadanimal.’

. ‘Practice with live animals is needed for the training.Simulation is not the same. Blood withdrawal wassimulated, then I had to go through a re-trainingprocess.’

5. Using live animals to overcome fear and stress whenhandling animals (emotional factor, 9% of totalresponses)

Some respondents provided personal arguments, inwhich they indicated a change of emotional state as aresult of the use of live animals (being more confident,more comfortable, more secure, less fear, less stress), bycontrast with general arguments for the use of liveanimals.

. ‘In my opinion it’s very important to get in touchwith animals during the course and to lose fear andget comfortable handling the animals.’

. ‘I had no experience with live animals before, thusthe practical part of the course was the most impor-tant and useful for me. Before the course I was notconfident, insecure to work with animals.’

. ‘At least it could help me broke the fear to touch myexperimental animal.’

6. Using live animals for the benefits of future research,and of career (forward-looking factor, 8% of totalresponses)

Some respondents pointed out the benefits whichusing live animals should have for future research –better handling reduces measurement errors and, there-fore, the variability of the measures which are detrimen-tal to research, and hence increases the quality ofresearch – and in fine to careers.

. ‘I think without the use of live animals in the course,I wouldn’t be able to perform animal experiments athigh quality.’

. ‘If the researcher would not work with animals onthe course before performing the experiments, thequality of the data would not be reliable and theanimal welfare would be compromised.’

Views on the 3Rs and the use of live animals

Finally, an overwhelming majority of respondents con-sidered themselves to be very or fairly well informedabout the 3Rs (96%, among them 42% ‘very well’)and believed that these principles were adequatelyapplied during the course with regard to the use oflive animals (97% agree). This general agreement didnot differ among the sociodemographic and academicgroups. However, some respondents expressed con-cerns in the related open question, which indicatedsome misunderstandings of the goal, organization andlegal framework of the course, i.e. they wished to sharean animal with other participants, reuse the animals, orreduce the number of animals by practising only whatwas necessary; whereas the course builds individualskills, reuses all animals and is, by law, general.

. ‘Because we used a lot of animal: 1 per person whilewe could have use one for 3 or 4 persons.’

. ‘The animals should have been used further for stu-dies or more courses.’

. ‘As far as I know, in any university/institute one canfind rat/mice labs where need to euthanize the rat/mice as the experiment required. When such casesare accessible, for training students, we should usethose opportunities rather than euthanizing 2–3animal only for training one student. Furthermore,such training should be extremely narrow designed.For example, if someone will not need to euthanizeanimals during her/his research or inject, she/heshould not euthanize and inject animals as thiscause is unnecessary for the animal.’

. ‘The practical course develops interesting manipula-tion and gives ideas about what is possible, but is not


often adapted to what we will practice later. We per-form manipulation and kill animals for nothing inthese cases. I think that smaller varied workshopswould be better.’

. ‘I was just really uncomfortable that mice and ratswe used will be killed for basically nothing (we justinject PBS in these animals and perform a standardanaesthesia). This is for me really a pity that theseanimals are killed afterwards. I know they can’t beused anymore in other research projects, but theywould deserve to be sent for adoption for example.’

Discussion

This study sought to describe scientists’ attitudestowards the LAS courses in Switzerland through anonline survey sent to four cohorts of participants inthe LAS courses run between 2010 and 2016.Although this was not the main aim of the study, theresults provided a glimpse into the professional pathsthat scientists working in this field could experience.Their careers show frequent turnover (shown by thehigh number of invalid email addresses 2–6 yearsafter attendance on the LAS course), involve frequentinternational work or study experience (66% in 2010,69% in 2012, 47% in 2014, and 46% in 2016), andsometimes involve periods of unemployment after theparticipants receive their PhDs (4% in cohort 2010 and2% in 2012).

The analyses showed a great diversity among parti-cipants in terms of gender, age, nationality, first degreedisciplines, occupation, etc. This diversity constitutes amajor hurdle for LAS course organizers and tutors whoneed to adjust and respond to changes in subgroup sizesfrom year to year. For illustrative purposes, the percen-tage of participants having a first degree in biologyvaried from 51–69% among the cohorts; the varianceof age remained high within each cohort – SD of 6.8 inthe 2010 cohort and 6.1 in the 2016 cohort; and 14.4%of participants were Asians in 2012 whereas this pro-portion was 7.1% in 2010.

Overall, the LAS course participants approved ofand were very satisfied with the various aspects of thecourse (87–93%). These figures were in line with theresults of the few foreign published studies.14,15

Having said that, analysis of the assessment of the sub-groups indicated that some of them showed slightlymore reservations than others (biologists or partici-pants with an Asian nationality were the least favour-able to the course). In the open questions, somerespondents (particularly biologists and Asian respon-dents) expressed their preferences for a course withmore practice and with content tailored more to theirprevious knowledge and to their future knowledge

needs. These results pose questions concerning the use-fulness and feasibility of less general LAS courses.

Two to six years after the course, 5% of respondentswere no longer performing animal manipulations in theiractual job, and 19% had not performed animal experi-mentation so far. These results raise some questions onthe compulsory nature of the course and on the mostappropriate timeframe to ensure its relevance. Finally, ofthose who still performed animal manipulation, 32%performed easy and complex handling in their currentjobs, which reinforces the importance of continuoustraining. Current FELASA recommendations involvethe need for institutions ‘to ensure that personnel arecompetent and trained continually’ (for at least oneday a year in Switzerland).17

One of the features of this study was its combinationof closed and open questions, and the latter materialwas very useful for interpreting the very positive feed-back from participants regarding the use of live animalsduring the course. The arguments provided by respon-dents cover areas as diverse as cognitive factors (i.e.related to practice, supervision and reality), emotionalfactors, and forward-looking (i.e. related to the welfareof animals used in, and the high-quality of, futureresearch) factors. The culture of care and animal wel-fare in animal experimentation that is learned in thiscourse falls within what Holmberg described as‘learning good handling as a means of doing goodresearch’.16 Among the cognitive factors, one factorrefers to the use of alternatives (simulations andvideos) in the course, and it seems that these alterna-tives cannot replace practice with live animals, which isessential for learning for some participants. Anotherinteresting argument indicates that not using live ani-mals during the course, in order to try to satisfy the 3Rswould result in real learning being only achieved afterthe course, which would result in increased animal suf-fering as there would be no one on site to teach andcorrect the researcher (less refinement), and which mayimply the further loss of animals (less reduction).Therefore, any gains achieved from applying the 3Rsduring the course would result in less refinement andreduction in the lab after the course for some respon-dents. Finally, the open question on the 3Rs indicatedthat some participants had not fully understood thegoal, organization and legal framework of the course.For example, some expressed a wish for the animals tobe reused, even though they are already reused. There isa need for LAS organizers and tutors to better explaintheir aims and choices related to the use of animalsduring the course. Sharing their reflections on thisissue is also a way of increasing participants’ awarenesstowards animals.

It shouldbe acknowledged that there are certain limita-tions to this study. First, with the method of sampling


chosen, we did not intend for this study to be representa-tive of the entire population of scientists doing animalexperimentation in Switzerland. We sampled amongcourse participants from within six years ago to ensurethey had a reliable memory of the course. The positiveassessment among the cohorts seems to highlight the con-stant effort of LAS teachers to improve their courses;however, this result should be confirmed more fully infuture years. Finally, there is a need for research on thisissue aimed at confirmed scientists with more experienceand at other stakeholders.

Second, the questionnaire did not distinguishbetween the two rodent species under consideration,but some participants made distinctions between miceand rats in the open questions, and the LAS organizersindicated some differences: there was some tendency forparticipants to be more apprehensive at working withrats in the beginning but to be more attached to theirrats at the end. A later study should address this aspect.

Conclusion

The 2010/63/EU Directive emphasizes the importanceof training and competence for scientists performinganimal experimentation.3 The animal experimentationfield is often described as performing ‘selective open-ness’18 towards those outside academia, but whatabout the level of openness within the field betweenscientists and those who make decisions for them? InSwitzerland, decisions are made after consultation withvarious representatives (i.e. from academic and indus-trial research, universities, animal protection and can-tonal authorities), but legislators rarely listen directly toconcerns and suggestions from scientists. The lack ofinformation from those concerned may complicate theimplementation of decisions. The study reported in thisarticle has given a voice to scientists who have to takethe course in Switzerland. Such a study should be repli-cated in other European countries because cultural andpolitical differences may be significant.

Acknowledgements

The author would like to thank Philippe Bugnon (LTK),Fabienne Chabaud-Barandum (ResAL), Marcel Gyger (EPFL)

for their inputs into the design of the research (i.e. email provi-sion) and their comments on a previous version of this article.




Funding

The author(s) received no financial support for the research,

authorship, and/or publication of this article.

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2011; 6: e22478.

12. van Luijk J, Cuijpers Y, van der Vaart L, et al. Assessing

the search for information on three Rs methods, and their

subsequent implementation: a national survey among

scientists in The Netherlands. ATLA 2011; 39: 429–447.

13. National Centre for the Replacement Refinement and

Reduction of Animals in Research (NC3Rs). Views on

the 3Rs – Survey Report 2008, www.nc3rs.org.uk/about-

us/corporate-publications (2008, last accessed April

2012).

14. Franco NH and Olsson IAS. Scientists and the 3Rs: atti-

tudes to animal use in biomedical research and the effect

of mandatory training in laboratory animal science. Lab

Anim 2013; 48: 50–60.


15. Carlsson HE, Hagelin J, Hoglund U, et al.

Undergraduate and postgraduate student’s responses to

mandatory courses (FELASA category C) in laboratory

animal science. Lab Anim 2001; 35: 188–193.16. Holmberg T. A feeling for the animal: on becoming an

experimentalist. Soc Anim 2008; 16: 316–335.

17. Guillen J. FELASA guidelines and recommendations.J Am Assoc Lab Anim Sci 2012; 51: 311–321. (Quotepage 315).

18. Holmberg T and Ideland M. Secrets and lies: ‘selectiveopenness’ in the apparatus of animal experimentation.Public Understanding Sci 2012; 21: 354–368.

Resume

Depuis 1999, la Suisse a mis en place une formation obligatoire dans le domaine de la science des animaux delaboratoire mais aucune evaluation complete de ses effets n’a ete entreprise jusqu’a present. Les resultats del’analyse des participants aux cours suisses obligatoires FELASA de categorie B en sciences des animaux delaboratoire en 2010, 2012, 2014 et 2016 ont montre que les participants ont pleinement apprecie tous leselements du cours. L’utilisation d’animaux vivants dans le cadre du cours a ete soutenue et expliquee par sixarguments invoquant des facteurs cognitifs, emotionnels et prospectifs. Une large majorite a estime que lesprincipes des 3R etaient correctement appliques pendant le cours. Les reponses a des questions ouvertes ontpermis de faire ressortir des idees d’ameliorations. Cette image positive globale revele cependant desreponses differenciees lorsque l’on considere les differentes sous-populations de notre echantillon (parexemple, les scientifiques ayant plus de recul, les scientifiques formes en biologie, les participants despays asiatiques).

Abstract

Obgleich in der Schweiz 1999 eine obligatorische Schulung in Versuchstierkunde eingefuhrt wurde, erfolgtebisher keine umfassende Auswertung ihrer Auswirkungen. Die Ergebnisse der Analysen von Teilnehmern anden Schweizer FELASA-Pflichtkursen (Kategorie B) in Versuchstierkunde in den Jahren 2010, 2012, 2014 und2016 zeigen, dass die Teilnehmer alle Elemente des Ausbildungskurses fur sehr wichtig hielten. Die Nutzungvon lebenden Tieren im Kurs wurde durch sechs Argumente unter Berucksichtigung kognitiver, emotionalerund zukunftsorientierter Faktoren unterstutzt und erlautert. Eine große Mehrheit meinte, dass die 3R-Prinzipien wahrend des Kurses angemessen berucksichtigt wurden. Antworten auf eine offene Frage botenIdeen fur Verbesserungen. Neben diesem positiven Gesamtbild ist jedoch auch auf die differenziertenAntworten zu verweisen, die von verschiedenen Teilgesamtheiten unserer Stichprobe erfasst wurden (zumBeispiel Wissenschaftler mit mehr nachtraglicher Einsicht, in Biologie ausgebildete Wissenschaftler,Kursteilnehmer aus asiatischen Landern).

Resumen

Suiza lleva implementado una formacion obligatoria en las ciencias de animales de laboratorio desde 1999,sin embargo, hasta el momento no se ha llevado a cabo una evaluacion a gran escala de su efectividad. Losresultados del analisis de participantes de los cursos obligatorios de categorıa B de FELASA Suiza de lasciencias de animales de laboratorio en 2010, 2012, 2014 y 2016 mostraron que los participantes apreciaronexhaustivamente todos los elementos del curso. El uso de animales vivos durante el curso fue respaldado yexplicado mediante seis argumentos que invocan factores cognitivos, emocionales y progresistas. Una granmayorıa considero que los principios de las 3 R fueron aplicados adecuadamente durante el curso. Distintasrespuestas a una pregunta abierta arrojaron ideas para conseguir mejoras. Sin embargo, esta vision generalpositiva revela respuestas diferenciadas al considerar distintas subpoblaciones en nuestra muestra (porejemplo, cientıficos con mas restrospeccion, cientıficos formados en biologıa o participantes de paısesasiaticos).


ab oratory

l i m i t e d

lan imals

Original Article

Quality of reporting of otorhinolaryngologyarticles using animal models with theARRIVE statement

Aren Bezdjian1,2,3, Sjaak F L Klis1,2, Jeroen P M Peters1,2,Wilko Grolman1,2 and Inge Stegeman1,2

AbstractResearch involving animal models is crucial for the advancement of science, provided that experiments aredesigned, performed, interpreted, and reported well. In order to investigate the quality of reporting of articlesin otorhinolaryngology research using animal models, a PubMed database search was conducted to retrieveeligible articles. The checklist of the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelineswas used to assess the quality of reporting of articles published in ear, nose and throat (ENT) and multidis-ciplinary journals. Two authors screened titles, abstracts, and full texts to select articles reporting otorhino-laryngology research using in vivo animal models. ENT journals (n¼ 35) reported a mean of 57.1% adequatelyscored ARRIVE items (median: 58.3%; 95% confidence interval [CI; 53.4–60.9%]), while articles published inmultidisciplinary journals (n¼ 36) reported a mean of 49.1% adequately scored items (median: 50.0; 95% CI[46.2–52.0%]). Articles published in ENT journals showed better quality of reporting of animal studies basedon the ARRIVE guidelines (P< 0.05). However, adherence to the ARRIVE guidelines is generally poor in oto-rhinolaryngology research using in vivo animal models. The endorsement of the ARRIVE guidelines byauthors, research and academic institutes, editorial offices and funding agencies is recommended forimproved reporting of scientific research using animal models.

Keywordsquality of reporting, animal studies, publication, otorhinolaryngology

Date received: 13 February 2017; accepted: 12 June 2017

Research involving animal models plays an importantrole in scientific innovation provided that the experi-ments are designed, performed, interpreted, andreported well. Recently, a lack of transparency inscientific publications using animal models has beenidentified.1 Crucial experimental design elements arefrequently ignored in scientific publications, renderingexperiments irreproducible.

Kilkenny et al. reported that 59% of investigated art-icles using animal models failed to adequately report aresearch hypothesis, objective, or the number and char-acteristics of the animals used (i.e. species/strain, sex,and age/weight).2 The vast majority of these articlesdid not report randomization of animals (87%) or blind-ing of researchers (86%). A similar study revealed that72% of articles using animal models did not report ran-domization of animals and 98% did not report blindingof researchers.3 Others have reported deficiencies in

reporting important methodological parameters thatrender experiments irreproducible.4 Inadequate report-ing greatly hinders the conclusions drawn in scientificpublications, especially when they cannot be reproduceddue to non-transparent methodology.5 Studies may infact be well-designed and well-conducted. However,

1Department of Otorhinolaryngology and Head & Neck Surgery,University Medical Center Utrecht, Utrecht, The Netherlands2Brain Center Rudolf Magnus, University Medical Center Utrecht,Utrecht, The Netherlands3Department of Experimental Surgery, Faculty of Medicine, McGillUniversity, Montreal, Quebec, Canada

Corresponding author:J P M Peters, MD, Department of Otorhinolaryngology, UniversityMedical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, TheNetherlands.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 79–87



sagepub.co.uk/


DOI: 10.1177/0023677217718862


when poorly reported they may lead to inaccurate inter-pretation and translation.6,7

In an attempt to optimize the overall quality ofreporting of scientific research using animal models,the ARRIVE (Animal Research: Reporting ofIn Vivo Experiments) guidelines were developed.8

These guidelines aim to improve the experimentaldesign, analysis, and reporting of research usinganimal models.

Using the checklist of the ARRIVE guidelines as ascoring tool, our primary aim was to evaluate the qual-ity of reporting of articles using animal models in oto-rhinolaryngology research. Furthermore, the quality ofreporting of articles published in ear, nose and throat(ENT) journals was compared with those published inmultidisciplinary journals.

Methods

Journal selection

The quality of reporting of articles which describeanimal experiments in otorhinolaryngology researchwas compared between two journal categories: ENTjournals and multidisciplinary journals. Based on ISIWeb of Knowledge impact factors (www.webofknow-ledge.com, date inspected: 12 June,2015), the five ENTjournals with the highest impact factors in 2012 wereselected: Ear & Hearing (Ear Hear), Journal of theAssociation for Research in Otolaryngology (JARO),Head & Neck – Journal for the Sciences andSpecialties of the Head and Neck (Head Neck),Hearing Research (Hear Res), and Audiology &Neurotology (Audiol Neurotol). None of these journalsimplemented the ARRIVE guidelines in the‘Instructions to Authors’ (date inspected: 12 June2015). The top five multidisciplinary journals in 2013were Nature, Science, Nature Communications (NatCommun), Proceedings of the National Academy ofSciences of the United States of America (PNAS) andScientific Reports (Sci Rep). Two journals (Nature andNat Commun) recommended the ARRIVE guidelineswhen documenting animal studies (date inspected:12 June 2015). The included journals and their impactfactors are summarized in Table 1.

Search strategy

A PubMed database search was conducted on June 12,2015 using four predefined filters. First, an adapted ver-sion of the ENT filter developed by the Cochrane ENTgroup was used to retrieve articles conducting researchin otorhinolaryngology.9 Second, a filter was appliedto only retrieve research using animal models.10

Subsequently, date restrictions were applied per journal

category to limit the amount of retrieved articles. Wesearched PubMed for articles published in ENT jour-nals in the year 2014. Since less otorhinolaryngologyrelated articles are published in multidisciplinary jour-nals, we searched for articles conducting animal experi-ments in otorhinolaryngology research published inmultidisciplinary journals from 2010 to 2014. It isimportant to note that the ARRIVE guidelines werefirst published in 2010. Thus, studies published in multi-disciplinary journals in 2010 might have been writtenprior to the publication of these guidelines. An analysiswas performed to investigate correlations between yearof publication and quality of reporting.

The complete search syntax with specific filtersis outlined in Supplemental digital content 1 (seehttp://journals.sagepub.com/doi/full/10.1177/0023677217718862 for all supplementary materials inthis article).

Study selection

Two authors (SFLK and JPMP) independentlyscreened titles, abstracts and full texts of the retrievedarticles and selected those reporting in vivo animalexperiments. To be considered for inclusion, studiesmust have assessed preclinical phases of diseases or dis-orders commonly treated by otorhinolaryngologists.Discrepancies between the two reviewers were discusseduntil consensus was reached.

Table 1. Impact factors of the top five ENT and multidis-ciplinary journals.

JournalImpactfactor*

ENT journals

Ear & Hearing (Ear Hear) 3.262

Journal of the Association for Researchin Otolaryngoloy (JARO)

2.952

Head & Neck (Head Neck) 2.833

Hearing Research (Hear Res) 2.537

Audiology & Neurotology(Audiol Neurotol)

2.318

Multidisciplinary journals

Nature 42.531

Science 31.477

Nature Communications (Nat Commun) 10.700

Proceedings of the National Academy ofSciences of the United States ofAmerica (PNAS)

9.809

Scientific Reports (Sci Rep) 5.078

*Source: ISI Web of Knowledge 2012, Journal Citations Reports(JCR) via www.webofknowledge.com (accessed on 12 June 2015).ENT: ear, nose and throat.


Scoring articles

To assess the quality of reporting of articles, twoauthors (AB and SFLK) independently scored articlesusing the checklist from the ARRIVE guidelines. Thechecklist contains 20 points, some with subsections (a,b, c or d). Subsections were considered as separateitems for scoring, yielding a total of 38 items. Twoitems on the ARRIVE checklist (10c and 15b) wereoptional and were rarely applicable. Therefore, tostandardize our assessment of quality of reporting inall articles, these two items were excluded from the ana-lysis. In total, 36 items were scored for each article.

Supplemental digital content 2 summarizes the scor-ing criteria per item. Articles were reviewed in order toextract all provided information. This included supple-mentary information; available online or in appendices.No more than five articles per journal category werescored consecutively to distribute possible learningeffects evenly across the two journal categories.

Inter-observer agreement

Cohen’s kappa value for inter-observer agreement wasevaluated to analyze discrepancies among the scorers.Cohen’s kappa was calculated for the complete dataset,and per item.

Data analysis

Descriptive statistics including median and mean scoresof adequately reported ARRIVE items were calculated.A two-tailed Mann–Whitney U-test for two independ-ent samples was used to evaluate significant differencesbetween the two journal categories. Chi-square analysiswas used to evaluate each ARRIVE item between thetwo journal categories. For the articles published inmultidisciplinary journals, a correlation between yearof publication and quality of reporting was investigatedusing Spearman’s rank correlation coefficient(Spearman’s rho).

Statistical tests were performed using the SPSS v20statistics package (IBM, Armonk, NY, USA).Statistical significance was set at 5%.

Results

Search and study selection

The combined search syntaxes (Supplemental digitalcontent 1) yielded 51 articles published in ENT journals,and 63 articles in multidisciplinary journals. Figure 1summarizes the search and study selection process.

Of the 51 articles retrieved from ENT journals, 11were not primary research articles and five did notinvolve in vivo animal experiments. Therefore, 35 articles

from ENT journals were included in the analysis (HearRes: n¼ 15, JARO: n¼ 11, Head Neck: n¼ 9).

Of the 63 articles retrieved from multidisciplinaryjournals, 18 were not related to otorhinolaryngologyresearch, five did not report on primary research andfour did not include in vivo animal experiments.Therefore, 36 articles were included in the analysis(PNAS: n¼ 24, Nature: n¼ 4, Nat Commun: n¼ 4,Sci Rep: n¼ 3, Science: n¼ 1). Eight articles were pub-lished in multidisciplinary journals that endorse theARRIVE guidelines. Six articles were published inmultidisciplinary journals in 2010, eight in 2011, eightin 2012, nine in 2013 and five in 2014.

The numbers of retrieved and selected articles perjournal are summarized in Table 2.

Overall quality of reporting scores

The 35 articles published in ENT journals reported amean of 57.1% adequately scored items (95% confidenceinterval [CI]: 53.4–60.9%; median: 58.3%). The 36 art-icles published in multidisciplinary journals reported amean of 49.1% adequately scored items (95% CI: 46.2–52.0%; median: 50.0%). The overall difference betweenthe journal categories was statistically significant (Mann–WhitneyU-test,P¼ 0.001), suggesting thatENT journalsadhered better to the ARRIVE guidelines.

For the articles published in multidisciplinary jour-nals, there was no statistically significant correlationbetween the year of publication and the number ofadequately reported ARRIVE items (P¼ 0.083).Moreover, there was no significant difference in thequality of reporting between the eight articles publishedin multidisciplinary journals that endorsed theARRIVE guidelines (Nature, Nat Commun), and the28 articles published in journals that did not endorsethe ARRIVE guidelines: 51.4% (95% CI: 45.6–57.2%,median: 54.2%) compared with 48.4% (95% CI: 45.1–51.7%, median: 50.0%), respectively.

Quality of reporting for specific items

When examining ARRIVE items separately, five items(6a, 7a, 9c, 14, 18b) were scored significantly higher inthe articles published in ENT journals (Figure 2,Table 3). These items assessed if the study reportedthe number of experimental and control groups (6a),information on the drug dose, site and route of admin-istration, and surgical procedure and equipment used(7a), welfare-related assessments and interventions car-ried out prior, during, or after experiments (9c), infor-mation on health status of animals prior to treatmentor testing (14) and study limitations (18b).

Several items were not adequately reported in bothjournal categories: 10 items were reported less

Bezdjian et al. 81

frequently than 20% in both journal categories(Figure 2). These items include the time of day whenexperiments were carried out (7b), the rationale behindthe choice of the specific anesthetic, its dose and routeof administration opted for (7d), information regarding

housing of animals (9a), sample size calculation (10b),allocation of the animals to groups (11a,b), methodsused to assess whether the data met the assumptionsof the statistical approach (13c), reporting of adverseevents (17a,b), and implications of the experimentalmethods or findings for the replacement, refinementor reduction of the use of animals in research (18c).

Inter-observer agreement

Out of a total number of 2556 scored items, 158 (6.1%)were scored differently. Cohen’s kappa value for inter-observer agreement was 0.87 (standard error¼ 0.10).A Cohen’s kappa score between 0.61 and 0.80 suggestsa good agreement between independent scorers.11

Cohen’s kappa value for inter-observer agreement peritem is presented in Supplemental digital content 3. Theinter-observer agreement was high for most items, andthere were no Cohen’s kappa values lower than 0.3.11

Discussion

The present study evaluated the quality of reporting ofscientific publications using animal models in otorhino-laryngology research. Articles published in ENT jour-nals adhered better to the ARRIVE guidelines thanarticles published in multidisciplinary journals.

Figure 1. Flow chart demonstrating study selection.ENT: ear, nose and throat.

Table 2. Retrieved articles by search and included articlesfollowing study selection.

Retrieved Included

ENT journals

Hear Res 26 15

JARO 13 11

Head Neck 11 9

Ear Hear 1 0

Audiol Neurotol 0 0

Total 51 35

Multidisciplinary journals

PNAS 39 24

Nature 13 4

Nat Commun 6 4

Sci Rep 3 3

Science 2 1

Total 63 36

ENT: ear, nose and throat.


Therefore, articles published in multidisciplinary jour-nals with high impact factors do not have a superioroverall quality of reporting in otorhinolaryngologyresearch using animal models. Similarly, MacLeodet al. have identified significantly fewer reporting ofrandomization in articles published in journals withhigh impact factors.12 Our findings are contrary toreports investigating the quality of reporting of rando-mized controlled trials13 and systematic reviews14 inotorhinolaryngology research, where ENT journalsunderperformed.

Interpretation of results

Although ENT journals showed better quality ofreporting, adherence to the ARRIVE guidelines is gen-erally poor in otorhinolaryngology research for bothjournal categories. Items such as choice of the specificanesthetic, dose and route of administration (7d) andinformation regarding the housing of animals (9a) wererarely (<20% of all studies) reported. This informationis essential for accurate replication of animal

experiments, as it may influence study outcomes.15

Prager et al. reported that housing and husbandryinformation of animals have the potential to influenceresponses of rodents, and thus alter study outcomes.16

Our findings also revealed that sample size calculationfor the number of animals chosen per group (10b) andallocation of the animals to groups (11a,b) were rarelyreported (<20% of all studies). These two items areessential for optimizing statistical design, and for ful-filling ethical obligations, as they aim to reduce poten-tial bias and the number of animals used in research.1,17

Articles published in multidisciplinary journals oftendescribed additional experiments alongside the animalmodel. As such, the animal experiment could have notbeen the primary focus of the study. Nevertheless, allmultidisciplinary journals included had a methodologysection containing information relating to the animalexperiments. These sections do not have word limitsthat may have justified the missing information.

Similar outcomes are found in other disciplines.Gulin et al. performed a quality assessment review ofanimal studies for Chagas disease by comparing studies

Figure 2. Adequately reported scores per item according to the ARRIVE guidelines.*Significant difference (chi2) between journal type.Abstr: abstract; Adv ev: adverse events; Alloc: allocation of animals; Backgr: background; Exp an: experimental animals;Basel: baseline data; Exp out: experimental outcomes; Exp proc: experimental procedures; Fund: funding; Gen: gener-alisability; H & H: housing and husbandry; Interpr: interpretation; Nb an: number of animals; Obj: objectives; Study des:study design; SS: sample size; Stats: statistical method.

Bezdjian et al. 83

Table

3.Proportionofadequately

reportedARRIVEitemsperjournalca

tegory.

Item:

1:

Title

2:

Abstract

3a:

Back

ground

3b:

Back

ground

4:

Objective

5:

Ethic

*6a:

Study

design

6b:

Study

design

6c:

Study

design

*7a:

Exp

proc

7b:

Exp

proc

7c:

Exp

proc

7d:

Exp

proc

8a:

Exp

an

8b:

Exp

an

9a:

H&

H

9b:

H&

H

*9c:

H&

H

Articlespublish

ed

inENTjournals

(n¼35)

69%

91%

100%

86%

91%

94%

83%

23%

89%

89%

9%

46%

17%

83%

49%

20%

20%

46%

Articlespublish

ed

inmultidisciplinary

journals

(n¼36)

56%

89%

100%

69%

83%

92%

53%

11%

72%

56%

3%

42%

11%

61%

69%

17%

25%

17%

Pvaluech

i2

(2-tailed)

0.330

1.000

NA

0.155

0.478

1.000

0.011*

0.220

0.135

0.003*

0.357

0.813

0.514

0.064

0.094

0.767

0.778

0.011*

Item:

10a:

SS

10b:

SS

11a:

Alloca

tion

11b:

Alloca

tion

12:Exp

outcome

13a:

Stats

13b:

Stats

13c:

Stats

*14:

Base

l

15a:

Nban

16:

Outcome

17a:

Advev

17b:

Advev

18a:

Interpretation

18b:

Interpretation

18c:

Interpretation

19:

Gen

20:

Fund

Articlespublish

ed

inENTjournals

(n¼35)

74%

0%

20%

11%

97%

89%

80%

14%

29%

71%

94%

20%

6%

97%

49%

9%

97%

97%

Articlespublish

ed

inmultidisciplinary

journals

(n¼36)

56%

6%

14%

3%

100%

78%

58%

11%

3%

86%

97%

6%

6%

100%

22%

3%

94%

100%

Pvaluech

i2

(2-tailed)

0.137

0.493

0.541

0.199

0.493

0.343

0.072

0.735

0.003*

0.155

0.614

0.085

1.000

0.493

0.026*

0.357

1.000

0.493

*Significantdifference

(chi2)betw

eenjournalca

tegories.

Advev:

adverseevents;Base

l:base

linedata;Gen:generalisa

bility;

Fund:funding;Exp

an:exp

erimentalanim

als;Exp

outcome:exp

erimentaloutcome;Exp

proc:

exp

erimentalproce

-dures;

H&

H:housingandhusb

andry;n:number;

Nban:numberofanim

als;SS:sa

mple

size;Stats:statisticalmethods.


published before and after the publication of theARRIVE guidelines. In line with our findings, theirstudy revealed that items such as randomization(16%) and sample size calculations (7%) were rarelyreported.18 Ting et al. investigated interventionalanimal studies in rheumatology and reported missinginformation such as randomization (17%), sample sizecalculation (0%), allocation (0%), housing, husbandryand welfare-related information (5%), and implicationsfor replacement, refinement or reduction of the use ofanimal assessments (0%).19 These items are essential toreduce bias in scientific research, and to make experi-ments transparent and replicable.20 Furthermore,Schwarz et al. reviewed publications on preclinicalresearch for the treatment of mucositis/peri-implantitis,Freshwater et al. conducted reviews on animal researchpublished in plastic surgery journals, and Tsilidis et al.investigated the reporting of animal models for neuro-logical diseases.21–23 All studies concluded that there isan urgent need for improving the quality of reportingwhen using animal models.

Methodological considerations

Strengths of our study include a search strategy thatcould be reproduced to evaluate the quality of report-ing of animal studies in other disciplines. To accountfor learning effects, the two authors who independentlyscored 2556 items did not score more than five articlesconsecutively per journal category. The limitations ofthe study include firstly a subjective assessment by thetwo independent scorers. The scorers were also notblinded to which journal category the paper belonged.However, the high inter-observer agreement demon-strated that both reviewers had fairly similar judgment(Supplemental digital content 3). Second, in order toobtain a sufficient amount of articles, we included art-icles published in multidisciplinary journals from 2010–2014, whereas we included articles published in ENTjournals in 2014 only. Since the ARRIVE guidelineswere first published in 2010, articles published thatyear could not have had access to these guidelines.Nevertheless, a subanalysis revealed no correlationbetween the year of publication and the quality ofreporting. A third limitation is that Nature and NatCommun have recommended that authors use theARRIVE checklist (date inspected: 12 June 2015).However, no statistical difference was found in thequality of reporting between articles published in multi-disciplinary journals that endorsed the ARRIVE guide-lines and those that did not. Finally, our findings wereonly based on eight journals (ENT journals: Hear Res:n¼ 15, JARO: n¼ 11, Head Neck: n¼ 9; multidisciplin-ary journals: PNAS: n¼ 24, Nature: n¼ 4, NatureComm: n¼ 4, Science Rep: n¼ 3, Science: n¼ 1).

Reporting guidelines

Evidence that clinical trials lacked crucial methodo-logical information led to the development of theConsolidated Standards for Reporting Trials(CONSORT) statement, which is now implementedby many journals and funding agencies. Implementingthe CONSORT statement has been shown to drastic-ally improve the quality of reporting of clinicaltrials.24–27 By contrast, the development of theARRIVE guidelines did not enhance quality of report-ing when comparing articles appearing before and afterthe ARRIVE guidelines were published.28 Baker et al.showed that reporting of animal research in PLoS jour-nals, which have been early proponents of the ARRIVEguidelines, still remained low.28 In our sample, we alsoshowed that there was no improvement in the quality ofreporting with increasing year of publication (2010–2014). Therefore, we recommend a stronger endorse-ment of the ARRIVE guidelines from authors, journaleditors and funding agencies.

Conclusion

Although articles using animal models published inENT journals have better quality of reporting scoresthan those published in multidisciplinary journals,adherence to the ARRIVE guidelines is generallypoor in otorhinolaryngology research. There is anurgent need to improve the quality of reporting in oto-rhinolaryngology research using animal models.Editorial endorsement of the ARRIVE guidelinesfrom authors, research and academic institutes, editor-ial offices, and funding agencies is warranted to opti-mize quality of reporting.




Funding

The author(s) received no financial support for the research,authorship, and/or publication of this article.

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analyses of otorhinolaryngologic articles based on the

PRISMA statement. PLoS One 2015; 10: e0136540.

15. Von der Behrens W. Animal models of subjective tin-

nitus. Neural Plast 2014; 2014: 741452.

16. Prager EM, Bergstrom HC, Grunberg NE and Johnson

LR. The importance of reporting housing and husbandry

in rat research. Front Behav Neurosci 2011; 5: 1–4.

17. Festing MF and Altman DG. Guidelines for the designand statistical analysis of experiments using laboratoryanimals. ILAR J 2002; 43: 320.

18. Gulin JE, Rocco DM and Garcia-Bournissen F. Qualityof reporting and adherence to ARRIVE guidelines inanimal studies for Chagas disease preclinical drugresearch: a systematic review. PLoS Negl Trop Dis

2015; 9: e0004194.19. Ting KH, Hill CL and Whittle SL. Quality of reporting

of interventional animal studies in rheumatology: a sys-

tematic review using the ARRIVE guidelines. Int JRheum Dis 2015; 18: 488–494.

20. Hirst JA, Howick J, Aronson JK, Roberts N, Perera R

and Koshiaris C. The need for randomization in animaltrials: an overview of systematic reviews. PLoS One 2014;9: e98856.

21. Schwarz F, Iglhaut G and Becker J. Quality of assess-ment of reporting of animal studies on pathogenesisand treatment of peri-implant mucositis and peri-implan-titis. A systematic review using the ARRIVE guidelines.

J Clin Periodontol 2012; 39: 63–72.22. Freshwater MF. Laboratory animal research published in

plastic surgery journals in 2014 has extensive waste: a

systematic review. J Plast Reconstr Aesthet Surg 2015;68: 1485–1490.

23. Tsilidis KK, Panagiotou OA, Sena ES, et al. Evaluation

of excess significance bias in animal studies of neuro-logical diseases. PLoS Biol 2013; 11: e1001609.

24. Plint AC, Moher D, Morrison A, et al. Does theCONSORT checklist improve the quality of reports of

randomised controlled trials? A systematic review. MedJ Aust 2006; 185: 263–267.

25. KaneRL,Wang J andGarrard J.Reporting in randomized

clinical trials improved after the adoption of theCONSORT statement. JClin Epidemiol 2007; 60: 241–249.

26. Altman DG. Endorsement of the CONSORT statement

by high impact medical journals: survey of instructionsfor authors. BMJ 2005; 330: 1056–1057.

27. Hopewell S, Altman DG, Moher D and Schulz KF.

Endorsement of the CONSORT statement by highimpact factor medical journals: a survey of journal edi-tors and journal ‘Instructions to Authors’. Trials 2008; 9:20.

28. Baker D, Lidster K, Sottomayor A and Amor S. Twoyears later: journals are not yet enforcing the ARRIVEguidelines on reporting standards for pre-clinical animal

studies. PLoS Biol 2014; 12: e1001756.

Resume

La recherche impliquant des modeles animaux est cruciale pour l’avancement de la science a condition queles experiences soient bien concues, realisees, interpretees et rapportees. Une recherche dans la base dedonnees Pubmed a ete effectuee afin de recuperer les articles admissibles pour enqueter sur la qualite desinformations rapportees par les articles de recherche otorhinolaryngologique utilisant des modeles animaux.La liste de verification des lignes directrices ARRIVE (de l’anglais Animal Research : Reporting of In VivoExperiments [Recherche animale : communication des experiences in vivo]) a ete utilisee pour evaluer laqualite des informations rapportees par les articles publies dans les journaux et revues multidisciplinairesd’ORL. Deux auteurs ont examine les titres, resumes et textes complets pour selectionner les


articles rapportant la recherche menee en otorhinolaryngologie sur des modeles animaux in vivo. Les revuesd’ORL (n¼ 35) montraient une moyenne de 57,1 % d’elements obtenant un score ARRIVE adequat (mediane :58,3 % ; intervalle de confiance a 95 % (IC) [53,4 - 60,9 %]), tandis que les articles publies dans les revuesmultidisciplinaires (n¼ 36), montraient une moyenne de 49,1 % d’elements obtenant un un score adequat(mediane : 50,0 ; IC a 95 % [46,2 - 52,0 %]). Les articles publies dans des revues d’ORL demontraient unemeilleure qualite des rapports d’etudes animales sur la base des lignes directrices ARRIVE (p< 0,05).Cependant, l’adhesion aux lignes directrices ARRIVE est generalement pauvre dans le secteur de larecherche en otorhinolaryngologie utilisant des modeles animaux in vivo. L’utilisation systematique deslignes directrices ARRIVE par les auteurs, les instituts universitaires et de recherche, les bureaux de redac-tion et les organismes de financement est recommandee afin d’ameliorer les rapports de recherche scien-tifique menee sur des modeles animaux.

Abstract

Forschung unter Einsatz von Tiermodellen ist fur den wissenschaftlichen Fortschritt von wesentlicherBedeutung, vorausgesetzt, die Experimente werden gut konzipiert, durchgefuhrt, interpretiert und berichtet.Zur Untersuchung der Berichts-Qualitat von Artikeln der HNO-Heilkunde-Forschung mittels Tiermodellenwurde eine PubMed-Datenbank-Suche zur Ermittlung relevanter Artikel durchgefuhrt. Anhand der Checklisteder ARRIVE (Animal Research: Reporting of In Vivo Experiments)-Richtlinien wurde die Berichts-Qualitat vonin HNO-Fachzeitschriften und multidisziplinaren Zeitschriften veroffentlichten Artikeln bewertet. ZweiAutoren sichteten Titel, Abstracts und Volltext zwecks Auswahl von Artikeln uber HNO-Forschung mittelsIn-vivo-Tiermodellen. HNO-Fachzeitschriften (n¼35) berichteten ein Mittel von 57,1 % adaquat bewerteteARRIVE-Elemente (Medianwert: 58,3 %; 95 % Konfidenzintervall (CI) [53,4 – 60,9 %]), wahrend in multidiszi-plinaren Zeitschriften erschienene Artikel (n¼36) ein Mittel von 49,1 % adaquat bewertete ARRIVE-Elementeberichteten (Medianwert: 50,0; 95 % CI [46,2 – 52,0 %]). In HNO-Fachzeitschriften veroffentlichte Artikelzeigten eine bessere Berichts-Qualitat von Tierstudien basierend auf den ARRIVE-Richtlinien (p<0,05).Dennoch ist die Einhaltung der ARRIVE-Richtlinien in der HNO-Heilkunde-Forschung mittels In-vivo-Tiermodellen generell durftig. Die Unterstutzung der ARRIVE-Richtlinien durch Autoren,Forschungseinrichtungen und wissenschaftliche Institute, Radaktionen und Finanzierungsstellen wird fureine optimierte Berichterstattung uber wissenschaftliche Forschung unter Einsatz von Tiermodellenempfohlen.

Resumen

La investigacion con modelos animales es crucial para el avance de la ciencia siempre que los experimentosesten bien disenados, realizados, interpretados y registrados. Para poder investigar la calidad del registro deartıculos en la investigacion de otorrinolaringologıa utilizando modelos animales, se realizo una busqueda enla base de datos Pubmed para recopilar artıculos elegibles. Se utilizo l lista de comprobacion de las direc-trices ARRIVE (Investigacion Animal: Registro de Experimentos In Vivo, por sus siglas en ingles) para evaluarla calidad del registro de artıculos publicado en revistas ENT y publicaciones multidisciplinarias. Dos autoresanalizaron tıtulos, resumenes y textos completos para seleccionar artıculos que registraran investigacionesde otorrinolaringologıa utilizando modelos animales in vivo. Revistas ENT (n¼35) arrojaron un promedio de57,1 % de artıculos ARRIVE adecuadamente puntuados (media: 58,3 %; 95 % intervalo de confianza (CI) [53,4 –60,9 %]), mientras que los artıculos publicados en revistas multidisciplinarias (n¼36) registraron un promediode 49,1 % de artıculos adecuadamente puntuados (media: 50,0; 95 % CI [46,2 – 52,0 %]). Los artıculospublicados en revistas ENT mostraron una mayor calidad del registro de estudios animales basado en lasdirectrices ARRIVE (p<0,05). No obstante, la adherencia a las directrices ARRIVE es por lo general baja en lainvestigacion de otorrinolaringologıa utilizando modelos animales in vivo. Se recomienda un respaldo a lasdirectrices ARRIVE por parte de autores, institutos academicos y de investigacion, oficinas editoriales yagencias de financiacion para un registro mejorado de la investigacion cientıfica utilizando modelos animales.

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Short Report

Ultrasound guided percutaneous commoncarotid artery access in piglets forintracoronary stem cell infusion

Jason H Anderson1, Nathaniel W Taggart1, Sarah L Edgerton3,Susana Cantero Peral4, Kimberly A Holst5 and Frank Cetta1,2;for the Wanek Program Preclinical Pipeline

AbstractIn pigs, the deep location of the common carotid artery and overlying sternomastoideus muscle in the neckhas led to the recommendation for a surgical cutdown for common carotid access, as opposed to minimallyinvasive techniques for vascular access. We sought to determine if direct percutaneous common carotidartery access in piglets is attainable. Seventeen piglets were anesthetized and intubated. Under two-dimen-sional and color flow Doppler ultrasound guidance, a 21 gauge needle was utilized to access the rightcommon carotid artery. Following arterial puncture, the Seldinger technique was applied to place a 4 or 5French introducer. Upon completion of cardiac catheterization with intracoronary stem cell infusion theintroducer was removed and manual pressure was applied to prevent hematoma development. Successfulaccess with an introducer was achieved in all 17 piglets. The average weight was 8.5� 1.7 kg. One pigletdeveloped a hematoma with hemorrhaging from the catheterization site and was euthanized. This piglet wasgiven bivalirudin for the procedure. After this incident, subsequent piglets were not given anticoagulation andno other complications occurred. Ultrasound guided percutaneous common carotid artery access in piglets isattainable in a safe, reliable, and reproducible manner when performed by microvascular experts.

Keywordsswine, ultrasonography, interventional, cardiac catheterization, carotid arteries

Date received: 27 April 2017; accepted: 18 June 2017

Dondelinger et al. documented a detailed overview ofradiological vascular and visceral anatomy of pigs.1

The right and left coronary artery origins lie in thebulb of the thoracic aorta cranial to the correspondingcusps of the aortic valve, similar to humans. Standardhuman diagnostic catheterization techniques andequipment have been utilized for coronary proceduresin adult swine.2 In piglets, the short length of theascending aorta limits the ability to safely and easilyengage the coronary ostia from a femoral arterialapproach using standard catheters available in theUSA. This difficulty is avoided by utilizing a commoncarotid artery approach that eliminates the need to tra-verse the aortic arch.

Historically, direct percutaneous catheterization ofthe common carotid artery has been avoided. Thedeep location and position of the common carotid

artery in relation to the sternomastoideus muscle inthe neck lead to the recommendation of a surgical cut-down for carotid access in pigs generally.1 Thus, wesought to evaluate the possibility of two-dimensional

1Division of Pediatric Cardiology, Mayo Clinic, Rochester, MN, USA2Department of Cardiovascular Medicine, Mayo Clinic, Rochester,MN, USA3Wanek Program for Hypoplastic Left Heart Syndrome, MayoClinic, Rochester, MN, USA4Division of General Internal Medicine, Mayo Clinic, Rochester, MN,USA5Department of Cardiothoracic Surgery, Mayo Clinic, Rochester,MN, USA

Corresponding author:Jason H Anderson, MD, Division of Pediatric Cardiology, MayoClinic, Rochester, MN 55905, USA.Email: [email protected]

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and color flow Doppler guided access techniques forpercutaneous access of the common carotid artery,obviating the need for a surgical cutdown.

Animals, materials and methods

Seventeen piglets aged 4–6 weeks were included in thisstudy. The piglets were offered no food, but did haveaccess to water, on the day of the operation.Intramuscular injections of telazol 5mg/kg andxylazine 1–2mg/kg were given for proceduralinduction. Hair removal over the ventral neck to theupper chest, soap wash and scrub, and peripheral intra-venous line placement were performed. Endotrachealintubation was achieved, and anesthesia was main-tained with 1–3% isoflurane. The surgical site wasscrubbed with a povidone–iodine solution andthen draped in a sterile fashion. Under ultrasound guid-ance using a SonoSite S Series device with a L25�probe (13–6mHz, 6 cm scan depth) (FUJIFILMSonoSite Inc, Bothell, WA, USA), a 21 gauge needlewas used to access the right-sided common carotidartery. The needle was advanced without an attachedsyringe thus allowing for pulsatile blood return onvessel entry. The needle was replaced with a 4 or 5French Terumo Pinnacle� Sheath (Terumo MedicalCorporation, Somerset, NJ, USA) using the Seldingertechnique.3 Anticoagulation with intravenous bivaliru-din 1mg/kg was used for the first case only. Thesubsequent 16 cases received no anticoagulation.Cardiac catheterization and direct right coronaryartery injection of contrast and stem cells were com-pleted using Optitorque� internal mammary orAmplatz left catheters (Terumo MedicalCorporation). After angiography and cell infusion, allcatheters and sheaths were removed by applyingmanual pressure over the vessel insertion site for10min. The site was monitored over the following120min and twice daily for 48 h after the procedure.

Our approach to vascular access is as follows:

1. The pig is placed in the supine position under generalanesthesia.

2. The manubrium is located with the ultrasound probeplaced laterally (the pig’s right side). The left side ofthe screen is to the pig’s right, confirming the medial/lateral orientation.

3. The probe is swept cranially and caudally on the skinto identify the bifurcation of the right subclavianfrom the bicarotid trunk (Figure 1).

4. The probe is directed 1–2 cm cranially following theright common carotid as it courses medially to theinternal jugular vein (Figure 2). Color flow Dopplerassists with vascular identification using this maneu-ver. Red color indicates flow coming toward the

probe (arterial with this orientation) and blue colorindicates flow away.

5. Theneedle is inserted at the centralmarkerof the probeat a 30� angle and advanced through the muscle to theartery. The needle tip is visible on ultrasound. Theartery depth is identified on the ultrasound, rangingfrom 2 to 2.5 cm below the skin with this population.

6. The right common carotid artery or bicarotid trunk ispunctured. Pulsatile blood flow from the hub of theneedle confirms an arterial source. A wire is advancedthrough the needle under fluoroscopic guidance. Thewire enters the ascending aorta and left ventricle orcourses down the descending aorta. If the wire divertsto the right heart border, the access is likely venousand the needle and wire are removed.

7. The needle is removed over the wire, and the hemo-static sheath is advanced.

8. The wire and sheath dilator are removed, and thesheath is flushed prior to cardiac catheterization.

9. When complete, the sheath is removed while apply-ing manual pressure over the vessel insertion site.

Figure 1. Two-dimensional with color flow Doppler ultra-sound image obtained in an axial plane at the level of themanubrium. The first brachiocephalic artery bifurcation isvisualized with anterior and rightward branching of thesubclavian artery (red color; asterisk). Medial to the bra-chiocephalic artery is the venous confluence forming theorigin of the superior vena cava (blue color; cross). A:anterior; P: posterior; R: right; L: left.

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All procedures were approved by the Mayo ClinicInstitutional Animal Care and Use Committee and con-formed to the National Institutes of Health guidelinesfor ethical animal research.

Results

Successful arterial access and subsequent cardiac cath-eterization were performed in all 17 piglets. The averageweight was 8.5� 1.7 kg. The average duration fromdraping the animal to completion of access was38.7� 15.5min. If vascular spasm or an inability topass a wire occurred, the needle was withdrawn andthe procedure restarted. The average procedural timefrom sedation onset to extubation was100.0� 28.8min. One piglet (1/17, 6%) developed ahematoma with hemorrhaging from the catheterizationsite and was euthanized. No other complications, suchas pneumothorax, vascular perforation, or stroke wereencountered.

Discussion

This study describes the successful utilization ofultrasound imaging for percutaneous common carotidaccess in young domestic swine. Overall, this is areliable, reproducible, and safe procedure for this popu-lation when performed by microvascular experts.Direct access was obtained in all animals, obviatingthe need for a surgical cutdown. One piglet waseuthanized due to hematoma development with hemor-rhaging from the access site immediately following lineremoval. This piglet was the first case and wasanticoagulated with bivalirudin, a direct thrombininhibitor. As a result of this complication, anticoagula-tion was not administered thereafter and no subsequenthemostatic complications occurred.

The utilization of ultrasound guided arterial accessis well established in humans. Seta et al. havereported a reduction in the number of attempts,time to vessel access, and inadvertent vessel accesswhen utilizing ultrasound guidance.4 Ultrasound ima-ging facilitates the analysis of vascular depth and sizewith localization of surrounding structures. Adetailed understanding of the vascular anatomy isnecessary for this procedure, and this has been pre-viously outlined.1

Acknowledgements

This work was supported by the Todd and Karen WanekFamily Program for Hypoplastic Left Heart Syndrome,

Mayo Clinic Foundation, Rochester, Minnesota, USA.Other members of the Wanek Program for Hypoplastic LeftHeart Syndrome Preclinical Pipeline who contributed to this

study were: Jennifer Miller, MBA, Joan Wobig, CSA, JoPowers, Scott Suddendorf, Saji Oommen, PhD andTimothy Nelson MD, PhD.




Funding

The author(s) disclosed receipt of the following financial sup-

port for the research, authorship, and/or publication of thisarticle: This study was funded by the Todd and Karen WanekFamily Program for Hypoplastic Left Heart Syndrome,

Mayo Clinic Foundation.

Ethical publication

The authors confirm that they have read the Journal’s pub-lication on issues involved in ethical publication and affirmthis report is consistent with those guidelines. This paper isnot under consideration elsewhere and none of the paper’s

contents have been previously published. All authors haveread and approved the manuscript.

Figure 2. Two-dimensional with color flow Doppler ultra-sound image obtained in an axial plane with the imagingprobe cranial to the manubrium. The trachea (doublecross) denotes the midline. The common carotid artery (redcolor; asterisk) courses anteriorly and is rightward of thetrachea. Anterior and lateral to the common carotid arteryis the internal jugular vein (blue color; cross). A: anterior;P: posterior; R: right; L: left.


References

1. Dondelinger RF, Ghysels MP, Brisbois D, et al.

Relevant radiological anatomy of the pig as a training model

in interventional radiology. Eur Radiol 1998; 8: 1254–1273.2. Suzuki Y, Yeung AC and Ikeno F. The representative

porcine model for human cardiovascular disease.

J Biomed Biotechnol 2011; 2011: 195483.

3. Seldinger SI. Catheter replacement of the needle in percu-

taneous arteriography; a new technique. Acta Radiol 1953;

39: 368–376.4. Seto AH, Abu-Fadel MS, Sparling JM, et al. Real-time

ultrasound guidance facilitates femoral arterial access

and reduces vascular complications: FAUST (Femoral

Arterial Access With Ultrasound Trial). JACC

Cardiovasc Interv 2010; 3: 751–758.

Resume

Etant donne l’emplacement profond dans le cou de l’artere carotide commune et du muscle sterno-cleido-mastoıdien la recouvrant chez les porcs, il a ete recommande de reduire l’acces a l’artere carotide communeet d’utiliser plutot les techniques d’acces peu invasives utilisees pour acceder a la veine jugulaire externe.Nous avons cherche a determiner si l’acces percutane direct a l’artere carotide commune chez les porceletsetait realisable. Dix-sept porcelets ont ete anesthesies et intubes. Une aiguille de calibre 21 a ete utilisee avecguidage echographique Doppler 2D en couleur pour acceder a l’artere carotide commune droite. Apresponction arterielle, la technique de Seldinger a ete utilisee pour placer un introducteur francais de 4 ou 5.Apres catheterisme cardiaque et injection intracoronaire de cellules souches, l’introducteur a ete retire parpression manuelle pour empecher la survenue d’un hematome. L’acces fructueux avec l’introducteur a eteatteint chez les 17 porcelets. Le poids moyen etait de 8,5� 1,7 kg. Un porcelet a developpe un hematomeaccompagne d’une hemorragie au site de catheterisme et a ete euthanasie. Ce porcelet a recu de la bivalir-udine pour la procedure. L’anticoagulation n’a pas ete utilisee apres ce cas et aucune complication ulterieurene s’est produite. L’acces percutane guide par ultrasons a l’artere carotide commune chez les porcelets estainsi possible de maniere sure, fiable, et de facon reproductible lorsqu’il est realise par des experts en accesmicrovasculaire.

Abstract

Bei Schweinen liegen Carotis-Arterie und der daruber befindliche Musculus sternocleidomastoideus tief imHals, weshalb fur den Zugang zur Carotis-Arterie generell eine chirurgische Freilegung anstelle der fur denZugang zur außeren Drosselvene angewandten minimal-invasiven Zugangsverfahren empfohlen wird.Wir wollten ermitteln, ob direkter perkutaner Carotis-Arterien-Zugang bei Schweinen moglich ist.Dazu wurden siebzehn Ferkel betaubt und intubiert. Eine 21-G-Kanule wurde mit Hilfe von 2D undFarbfluss-Doppler-Sonografie fur den Zugang zur rechten Carotis-Arterie verwendet. Nach arteriellerPunktion wurde mittels Seldingermethode ein 4 - oder 5-Seldingerdraht eingebracht. Nach erfolgterHerzkatheteruntersuchung mit intrakoronarer Stammzelleninfusion wurde der Draht unter manuellerDruckanwendung zwecks Verhinderung eines Hamatoms entfernt. Bei allen 17 Ferkeln war der Zugangmittels Katheter erfolgreich. Ihr Durchschnittsgewicht betrug 8,5� 1,7 kg. Ein Ferkel, bei dem einHamatom mit Blutung aus der Katheterisierungsstelle auftrat, wurde eingeschlafert. Dieses Ferkel erhieltBivalirudin fur den Eingriff. Nach Auftreten dieses Fall wurde auf Antikoagulation verzichtet und es kamzu keinen weiteren Komplikationen. Perkutaner Carotis-Arterien-Zugang unter Einsatz von Sonografie beiFerkeln kann sicher, zuverlassig und reproduzierbar erfolgen, sofern der Eingriff von Mikrovaskular-Spezialistendurchgefuhrt wird.

Resumen

En cerdos, la profunda ubicacion en el cuello de la arteria carotida comun y el musculo esternomastoideosuperpuesto hace que se recomiende un recorte quirurgico del acceso comun a la carotida, en lugar de lastecnicas de acceso invasivas mınimas utilizadas para llegar a la vena yugular externa. Tratamos de determi-nar si se puede conseguir un acceso a la arteria carotida comun percutanea en lechones. Se anestesiaron yentubaron diecisiete lechones. Se utilizo una aguja de calibre 21 bajo guıa ultrasonido Doppler a color y 2Dpara acceder a la arteria carotida comun derecha. Tras la puncion arterial, se utilizo la tecnica Seldinger paracolocar 4 o 5 introductores franceses. Una vez finalizada la cateterizacion cardıaca con infusion de celulas

Anderson et al. 91

madre intracoronaria, se extrajo el introductor con presion manual para evitar la aparicion de hematomas. Seconsiguio un acceso exitoso con un introductor en todos los 17 lechones. El peso medio era de 8,5� 1,7kilogramos. Un lechon desarrollo un hematoma con hemorragia en el lugar de la cateterizacion y fuesacrificado. Este lechon recibio bivalirudina para el procedimiento. No se utilizo la anticoagulacion despuesde este caso y no hubo ninguna otra complicacion. El acceso a la arteria carotida comun percutanea medianteultrasonido puede conseguirse de forma segura, fiable y reproducible cuando es realizado por expertosmicrovasculares.


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Case Report

Preputial diverticulum dilationin a Goettingen minipig

Nora Denk, Claudia Senn, Bjoern Jacobsen and Tobias Schnitzer

AbstractPreputial dilation is an infrequently reported condition in pigs. The pathophysiology and etiology is unclear.Causes for diverticulum dilation are proposed to be chronic preputial diverticulitis with subsequent fibrosis ofthe preputial cavity, phimosis of the preputial orifice or the preputial diverticulum, but the large majority ofcases are reportedly idiopathic in nature. Surgical interventions include ablative procedures, but many casesare not treated because of an assumed lack of clinical relevance in pigs not used for breeding. We report a caseof progressive preputial dilation that recurred after surgical intervention. Histopathological examinationrevealed no primary inflammatory condition, contrary to literature suggesting a role for inflammatory mediatorsin pathogenesis. Phimosis of the preputial orifice was noted post mortem and might be a contributing factor.

These findings partially contradict the current assumptions in regards to pathophysiology and treatmentchoices in the literature and warrant further investigation into alternative therapeutic interventions for thiscondition.

Keywordsmammal, organisms and models, pathology, physiology, reproduction, surgery, techniques

Date received: 26 January 2017; accepted: 14 September 2017

Case report

Single case reports of dilation of the preputial sachave been reported in boars, most often beingobserved during puberty. Affected pigs may beunsuitable for breeding purposes as the erect penismight get stuck in the dilated diverticula.1 The pre-putial cavity of the boar is significantly longer thanthe intrapreputial part of the penis. The preputialdiverticulum is a pear-shaped, bi-lobed recess of theanterior preputial cavity. Its neck, which is 1 cm to2 cm in diameter, opens onto the dorsal aspect of theprepuce, 3 cm to 4 cm caudal to the preputial ostium.The boar is the only domestic animal with a diver-ticulum. Normal contents of the diverticulum includeurine, semen, desquamated epithelial cells, bacteria,and occasional concretions. Urination flushes thediverticulum. During breeding, preputial muscle activ-ity causes the contents of the diverticulum to beexpelled. This results in semen contamination, apotentially significant problem in swine used for arti-ficial insemination.1

Reported causes for diverticulum dilation arechronic preputial diverticulitis and consecutive fibrosisof the preputial cavity, phimosis of the preputial orificeor the preputial diverticulum, but the large majoritywere reportedly idiopathic in nature. Surgical preputialdiverticulum ablation or no intervention at all due tono assumed clinical relevance in pigs not used forbreeding purposes are being recommended in the litera-ture. A method for surgical correction was published,which is also being used for other indications.Extirpation and ablation of the preputial diverticulumin pigs has been described for various reasons such asreduction of odor in male pet pigs, aesthetics, to relieveor prevent infection of the tissue, for decreasing therisk of contamination when collecting sperm for

Pharma Research and Early Development (pRED), PharmaceuticalSciences (PS), Roche Innovation Center Basel, Switzerland

Corresponding author:Nora Denk, F Hoffmann-La Roche AG Grenzacherstrasse 124Basel, 4070 Switzerland.Email: [email protected]

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reproductive purposes, and/or for treatment of preputialdiverticulum dilation. Three different methods have beendescribed: removal of the diverticulum via a paramedianincision lateral to the prepuce, or extirpation and excisionof either only the diverticular horns or the entire diver-ticulum through the preputial orifice.1–4 All are con-sidered to be minimally invasive procedures with littlepotential for complications. However, to our knowledgeno study has followed up pigs consecutively treated.

We report here a case of a male Goettingen minipigwith progressive preputial dilation and urine retention.This minipig was treated with partial diverticulumresection. We describe clinical outcomes, macroscopicand histopathological findings, and compare those to ahealthy age-matched control minipig.

The case

A 5.5-month-old male intact minipig (previouslyobtained from Ellegaard Goettingen Minipigs ApS,Dalmose, Denmark) with no previous medical historywas noticed to show a slightly increased volume ofapproximately 5 cm of the preputial sac comparedto other age-matched animals (approximately 2 cm to3 cm). Over the course of four weeks, this increased fur-ther in size, reaching a volume of approximately 9 cm indiameter (Figure 1) and containing about 250ml of urine.

The urine accumulation had to be manually squeezedout once daily, before surgical intervention. Even thoughthis was performed gently, the animal showed increasingdiscomfort and distress when manipulated. Under gen-eral anesthesia (ketamine 20mg/kg intramuscularly(IM), xylazine 2mg/kg IM) an ultrasound of the uro-genital region was performed that revealed no abnorm-alities other than dilated diverticulum horns.Ultrasound-guided cystocentesis allowed sterile collec-tion of urine for urinalysis, which was without abnormalfindings. Manual palpation revealed only a fairly smallopening to the posterior preputial cavity that was largeenough to insert a fingertip of a little finger. Using arubber tube attached to a 50 cc syringe, negative pressurewas applied to the penis tip and we attempted to pro-trude it from the preputial cavity. However, only thevery tip could shortly be visualized through the preputialorifice and it was not possible to advance the penis anyfurther or grasp it with tissue sponges.

The area was prepped in a sterile manner and draped,and the preputial cavity was flushed with 1% dilutedpovidone iodine solution prior to surgery. The diverticu-lum pouches were exteriorized one at a time by insertingAllis forceps into the preputial opening and directingthem laterally. After grasping the lining with the forceps,the lining was steadily pulled out of the opening. Onlythe ends of the horns could be exteriorized. The hornswere clamped and fixated with curved hemostatic

forceps, the horns excised, and the remaining tissuewas sutured with single interrupted sutures (Vicryl 4–0)and repositioned (Figure 2). The resected tissue was sub-mitted for histopathological analysis that revealed noabnormal findings.

Postoperative analgesia was provided (meloxicam0.4mg/kg subcutaneously (SQ), q24 hours) for threeconsecutive days and the animal recovered smoothlyand uneventfully and only a minor swelling of the peri-preputial area was observed for two to three days.Immediately after surgery, normal urination and nourine retention was observed for the following threemonths. Minor bloody discharge was noted the two con-secutive days and resolved without further intervention.Consecutively, eight weeks after the surgery and com-plete recovery, the minipig was included in a terminalpharmacokinetic study with one intravitreal administra-tion of a compound and a follow-up time of one week.

During that study or three months post-surgery, amostly unilateral increase in volume of the preputialcavity with consecutive urine retention was observedsuggesting recurrence (Figure 3).

As the animal was at that point scheduled foreuthanasia and necropsy because of the terminalstudy design four weeks later, no further therapeuticinterventions were undertaken. During necropsy ofthe animal, genital tissue (penis within the preputialsac) was sampled for macroscopical and histopatho-logical examination from the affected animal aswell as from an age-matched control animal.Macroscopically, the direct preputium cranial of thepenis tip showed only a small passable opening of3mm to 4mm (in contrast to 10mm in the age-matched

Figure 1. Dilated preputial cavity with urine retention in a5-month old Goettingen minipig.


control). Nevertheless, the penis was freely movablewithout any malformations or other abnormalities.The left portion of the diverticulum pouch had a diam-eter of 1 cm and the right portion of 3 cm (symmetric

2.5mm in diameter in age matched control) and theepidermis exhibited a greenish color. Histologically,the epidermis of the diverticulum and the preputiumwas regularly keratinized. In the underlying dermiswas a moderate chronic histiocytic inflammation withpigmented macrophages (pigment consistent withhemosiderin confirmed by Prussian blue stain;Figure 4) corresponding to the greenish discolorationobtained macroscopically; this finding was consideredto be the residue of the surgical intervention with asso-ciated hemorrhage rather than part of the primarycause of diverticulum dilation. Additional minimalfocal subepidermal follicular aggregates were con-sidered within the normal variation since they were pre-sent in the age-matched control as well.

Discussion

To the best of our knowledge, this is the first reportedcase of preputial dilation in a Goettingen minipig in alaboratory animal setting and in general of recurrenceafter surgical correction of dilation of the diverticula ina boar. Based on the number and year of publication of

Figure 2. Surgical resection of the preputial diverticula (schematic drawing).

Figure 3. Recurrence of clinical symptoms unilaterallythree months after surgical intervention.

Denk et al. 95

the already published cases, it appears to be a ratherrare condition in the Goettingen minipig.

The pathophysiology of the observed findings isunclear. One could consider a primary phimosis of theorifice between the cranial and caudal part of the prepu-tium, resulting in a change in dynamics during urination,which may have resulted in consecutive dilation of thediverticula. This would also explain the recrudescencesince the phimosis itself was not corrected. Contrary topreviously reported assumptions and case reports, wedefinitively ruled out chronic posthitis or neoplasticchanges of the preputium as a primary cause. Notably,acute posthitis was observed only after surgery and waslikely unrelated to the diverticula resection. Also, resec-tion of the diverticula alone did not result in a successfullong-term management of the issue. For future casessimilar to the one described here, different surgical inter-ventions may result in improved clinical outcomes.For example, a removal of the diverticular horns in com-bination with a surgical correction of the phimosis of theorifice between the cranial and caudal portions of thepreputial cavity might provide recurrence-free diseaseresolution. Removal of the entire cranial portion ofthe preputium together with the horns, similar to the pro-cedure described by Dutton et al.,3 in combination withdilation and reconstruction of the preputial orifice, mayconstitute another solution.

In the context of laboratory animal medicine, ani-mals need to be monitored for this condition, despitethe rare occurrence. As a treatment strategy for thiscondition, short-term management with daily manualextrusion of urine can provide symptomatic relief whileassessing long-term therapeutic options. However, ifthe animal needs to be housed or included in a studyfor a prolonged duration exceeding several weeks, sur-gical management is inevitable given that the animalreported here showed clear aversion against the persist-ent required manipulation of the preputium.

Surgical intervention as described here providedthree months of relief, but did not prevent recurrencein this case. Therefore a surgical method different fromthe one described here would be advised, although thespecific approach to be applied requires further inves-tigation. In the current case, no effect on the study con-ducted was observed, although it would not berecommended to include such an animal in a studywith urogenital parameters (such as urine, sperm, etc.)as study endpoints. Since a potential genetic predispos-ition cannot be ruled out, affected animals should notbe used for breeding and the original breeding facilityshould be notified.


The author(s) declared no potential conflicts of interest withrespect to the research, authorship, and/or publication of this

article.

Funding

The author(s) received no financial support for the research,authorship, and/or publication of this article.

References

1. Plonait H. Erkrankungen und Operationen an den

Fortpflanzungsorganen des Ebers. In: Waldmann KH

(ed.) Lehrbuch der Schweinekrankheiten. Stuttgart:

Parey, 2001, pp.525–548.2. Swindle M. Urinary system and adrenal glands.

In: Swindle M (ed.) Swine in the laboratory. South

Carolina: CRC Press, 2007, pp.157–174.3. Dutton DM, Lawhorn B and Hooper RN. Ablation of the

cranial portion of the preputial cavity in a pig. J Am Vet

Med Assoc 1997; 211: 598–599.

4. Kross SB, Ames NK and Gibson C. Extirpation of the

preputial diverticulum in a boar. Vet Med Small Anim

Clin 1982; 77: 549–553.

Figure 4. Diverticulum of a minipig, regularly keratinized epidermis of the diverticulum and histiocytic inflammation withpigmented macrophages in underlying dermis (pigment consistent with hemosiderin confirmed by Prussian blue stain),(a) hematoxylin and eosin stain, (b) Prussian blue stain.


Resume

La dilatation du prepuce est une condition rare chez les porcs. La physiopathologie et l’etiologie de cettecondition ne sont pas clairement etablies. La dilatation du diverticule serait causee par une diverticulitepreputiale chronique avec fibrose subsequente de la cavite preputiale, phimosis de l’orifice ou du diverticulepreputial, mais la grande majorite des cas serait de nature idiopathique. Les interventionschirurgicales comprennent les procedures ablatives, mais de nombreux cas ne sont pas traites en raisond’un suppose manque de pertinence clinique chez les porcs non utilises pour l’elevage. Nous rapportons uncas de dilatation preputiale progressive qui est reapparu apres une intervention chirurgicale. L’examenhistopathologique n’a revele aucune condition inflammatoire primaire, contrairement a la litterature quisuggere l’implication des mediateurs inflammatoires dans la pathogenese. Le phimosis de l’orifice preputiala ete remarque post mortem et pourrait etre un facteur contributeur.

Ces resultats contredisent en partie les hypotheses actuelles en ce qui concerne la physiopathologie et letraitement de choix dans la litterature et indiquent qu’une investigation plus approfondie sur les autresinterventions therapeutiques traitant cette condition serait utile.

Abstract

Praputialdilatation ist ein Zustand, der selten bei Schweinen gemeldet wird. Pathophysiologie und Atiologiesind unklar. Als Ursachen fur Divertikeldilatation werden teilweise chronische Praputialdivertikulitis mitnachfolgender Fibrose der Praputialhohle, Phimose der Praputialoffnung oder des Praputialdivertikels ange-fuhrt, doch der uberwiegende Teil der Falle ist Berichten zufolge idiopathischer Natur. Zu operativenEingriffen gehoren ablative Verfahren, doch werden viele Falle aufgrund eines angenommenen Mangelsklinischer Relevanz von nicht zu Zuchtzwecken verwendeten Schweinen nicht behandelt. Wir berichten hiervon einem Fall progressiver Praputialdilatation, die nach einem operativen Eingriff auftrat. Die histopatho-logische Untersuchung ergab keinen primaren entzundlichen Zustand – im Gegensatz zu in der Literaturvertretenen Ansichten, die von einer Rolle entzundlicher Mediatoren in der Pathogenese ausgehen. Phimoseder Praputialoffnung, die post mortem festgestellt wurde, konnte einen Einfluss haben.

Diese Erkenntnisse widersprechen teilweise den aktuellen Aussagen zu Pathologie undBehandlungsmoglichkeiten in der Literatur und rechtfertigen weitere Untersuchungen alternativer therapeu-tischer Interventionen fur diesen Zustand.

Resumen

La dilatacion prepucial es un trastorno registrado con poca frecuencia en cerdos. La patofisiologıa y etiologıano estan claras. Las causas de la dilatacion del divertıculo se proponen que son una diverticulitis prepucialcronica con fibrosis posterior de la cavidad prepucial, fimosis del orificio prepucial o el divertıculo prepucial,pero la gran mayorıa de casos son registrados como idiopaticos por su tipo. Las intervenciones quirurgicasincluyen procedimientos ablativos, pero muchos casos no se tratan debido a una presunta falta de relevanciaclınica en cerdos no utilizados para la crıa. Registramos un caso de dilatacion prepucial progresiva quereaparecio despues de una intervencion quirurgica. El examen histopatologico revelo que no habıa ningunproblema inflamatorio principal, al contrario de lo que diversos estudios que sugieren una funcion para losmediadores inflamatorios en en la patogenesis. Se observo una fimosis del orificio prepucial post mortem ypodrıa tratarse de un factor contribuyente.

Estas averiguaciones contradicen en parte las asunciones actuales sobre la patofisiologıa y las eleccionesde tratamientos de diversos estudios y avalan la realizacion de una investigacion adicional sobre interven-ciones terapeuticas alternativas para este problema.

Denk et al. 97

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News

Reorganization of the Journal’s EditorialBoard to Improve the Journal’sProductivity and Efficiency

Beat M Riederer1, Paulin Jirkof2 and Arieh Bomzon3

The rise of the journal’s impact factor and a concomi-tant increase in the number of submissions have made itnecessary to reorganize the journal’s editorial board(EB). The Journal has already re-organized its editorialteam1, and over the past months, the editorial boardwas re-organized and complemented with newmembers.

After many years of contributing to the journal’seditorial activities, several section editors left the EBfor different reasons. We thank Sheilah Robertson,Peter O’Brien, and Melissa Bateson for all their workand wish them success in their future endeavors andactivities. At the end of 2017, Nathalie Baehler, thejournal’s editorial assistant, left the editorial team.Initially, Nathalie was responsible for the first screenof incoming manuscripts which included checking foradherence to the ARRIVE reporting guidelines andover the past years she had oversight of the News sec-tion. We thank for her work and wish her all the best inher future activities.

Recently, the EB has undergone profound changes:several sections have been amalgamated, new sectionshave been created, and the number of editors has beenincreased in order to reduce the workload of the exist-ing section editor(s). Specifically, the ‘‘Anesthesia,Analgesia, Pain and Stress’’ section has been strength-ened with the addition of Patricia Foley and PatriciaHedenqvist. The ‘‘Anatomy and Neuroscience’’ sec-tion has been strengthened with the addition of SaraWells. The ‘‘Behaviour’’ section has been strength-ened with the addition of Marcel Gyger and LarsLewejohann. The ‘‘Biostatistics’’ section wasexpanded into a new ‘‘Biostatistics andExperimental Design’’ section and strengthened withthe addition of Hanno Wurbel. The ‘‘Education’’ sec-tion has been strengthened with the addition ofChrista Thone-Reinecke. The ‘‘Imaging Techniques’’section has been strengthened with the addition ofJordi Tremoleda. The ‘‘Physiology and ClinicalChemistry’’ section has been strengthened with theaddition of Tertius Hough. The ‘‘Primate’’ section

has been strengthened with the addition of PaulHoness. The ‘‘Reproductive Biology’’ has beenstrengthened by the addition of Colin Gilbert, a long-standing editorial board member, and Belen Pintado.The ‘‘Small Animal Models’’ section was strength-ened with the addition of Jan-Bas Prins and SaraWells. The ‘‘Systematic Review’’ section has beenstrengthened with the addition of Birgitte Kousholt.The ‘‘Veterinary Medicine’’ section has been strength-ened with the addition of NikolaosKostomitsopoulos. Two new sections were created:the ‘‘Aquatic Organisms’’ section whose editors areKarin Finger-Baier, Jean-Philippe Mocho andMarcus Crim, and the ‘‘Nutrition and Diets’’ sectionwhose editor is Graham Tobin. Several sections havebeen amalgamated, i.e. ‘‘Large Animal Models’’,‘‘Management of Animal Facilities’’, ‘‘Molecularand Genetic Engineering’’, ‘‘3Rs and Ethics’’, andthe editors of these amalgamated sections are long-standing members. The ‘‘Pathology andMicrobiology’’ section is an amalgamated sectionwhich has been strengthened with the addition ofAndre Bleich. The ‘‘Toxicology’’ and ‘‘SurgicalProcedures’’ sections may still need strengthening.Some of the new section editors have already startedhandling manuscripts. We would like to welcome allnew section editors and thank them for accepting towork for the Journal. At the same time, we also would

1Editor-in-Chief, Platform of Morphology, University of Lausanneand Centre for Psychaitric Neuroscience, University Hospital of theCanton Vaud, Lausanne, Switzerland2Deputy-Editor, Division of Surgical Research, University HospitalZurich, University of Zurich, Sternwartstr. 6, CH-8091 Zurich,Switzerland3Deputy-Editor, Consulwrite/Consulvet, 26 Hashoftim Street,Pardess Hanna-Karkur, 3707426, Israel

Corresponding author:Beat M Riederer, Platform of Morphology, University of Lausanneand Centre for Psychaitric Neuroscience, University Hospital of theCanton Vaud, 1005 Lausanne, Switzerland.Email: [email protected]

Laboratory Animals

2018, Vol. 52(1) 101–102



sagepub.co.uk/


DOI: 10.1177/0023677217752780


like to thank the longstanding section editors for alltheir hard work over the past years.

A special issue on Microbiota whose guest editorsare Axel Kornerup-Hansen and Craig Franklin isbeing planned for 2019 (date to be confirmed), andthey are actively recruiting contributions. We are look-ing forward to publishing some exciting minireviews

how microbiota are influencing laboratory animalexperiments.

Reference

1. Jirkof P, Riederer BM, Bomzon A. Update from the edi-tors. Laboratory Animals 2017; 51: 323.


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Obituary

Professor Klaus Gartner, 1927–2017

We note with sadness the passing of Klaus Gartner. Hewas one of the founding fathers/pioneers of laboratoryanimal science in Germany. Born in Pirna, Saxony,Germany, on 7 April 1927, he grew up in Pirna,Dresden, Magdeburg and Berlin, and graduated fromHumboldt University Veterinary School. Upon obtain-ing his doctorate (Dr. med. vet.), he initially joined theClinic for Small Animals at Humboldt University, inEast Berlin at that time, specializing in diseases of com-panion animals. Due to political incongruities, he hadto leave his university position in 1958. He left EastBerlin and joined the Institute of Physiology atHannover Veterinary University. Thereafter, in 1964,he was appointed head of an Animal ResearchFacility to be established at Frankfurt MedicalSchool. During all this time, his focus was directed atexperimental pathophysiology, in particular kidneyfunction, carbohydrate metabolism (diabetes), over-crowding and stress. Finally, Klaus Gartner wasappointed professor and director of the Institute ofLaboratory Animal Science and the Central AnimalFacility of Hannover Medical School, an institutionwhich he built up with great knowledge and emphasisand which was one of the first leading animal researchfacilities in Germany. More than 100 publications inscientific journals and handbooks show the extentof his clinical and experimental research in nephrology,physiology, endocrinology and laboratory animal

science. His achievements in laboratory animal scienceare multifaceted. He promoted the foundation ofthe Gesellschaft fur Versuchstierkunde (Society ofLaboratory Animal Science) (GV-SOLAS), the firstlaboratory animal science association in Europe, forwhich he served as honorary secretary during its firstyears in 1964–1969. He was member and co-chair ofthe Commission for Laboratory Animal Research ofthe German Research Council. He served as a reviewerfor many research projects, as a consultant for govern-mental and science organizations, and as a protagonistfor freedom of research, publicly promoting ethicallybased animal research and welfare. From 1973 on, hecoordinated the research unit ‘SFB 146, LaboratoryAnimal Research’ with three funding periods of threeyears and 15 to 20 projects funded in parallel, with theaim of establishing quality criteria for laboratoryanimal science (reported under ISBN 3-527-27717-X).Over the course of his research career, Klaus Gartnerestablished a national and international reputation andwas awarded several recognitions, for example honor-ary professorship at Hannover Veterinary Universityand Ludwig-Schunk-Award from Giessen University.He will be sadly missed as counsellor, mentor, sup-porter and open-minded audience.

Andre Bleich and Hans-Jurgen HedrichNovember 2017

Laboratory Animals

2018, Vol. 52(1) 105



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DOI: 10.1177/0023677217745777


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