The International Journal of Laboratory Animal Science, Medicine, Technology and Welfare

Volume 52 Number 4 August 2018 ISSN 0023-6772

Laboratory Animals THE INTERNATIONAL JOURNAL OF LABORATORY ANIMAL SCIENCE, MEDICINE, TECHNOLOGY AND WELFARE

Offi cial Journal of AFSTAL, ECLAM, ESLAV, FELASA, GV-SOLAS, ILAF, LASA, NVP, SECAL, SGV, SPCAL

Published on behalf of Laboratory Animals Ltd by SAGE Publications Press Ltd http://journals.sagepub.com/home/lan

laboratory an imals Volume 52 Number 4 August 2018 limited Contents

Review Article On determining sample size in experiments involving laboratory animals 341 MFW Festing

Original Articles Analgesics promote welfare and sustain tumour growth in orthotopic 4T1 and B16 mouse cancer models 351 J Lofgren, AL Miller, CCS Lee, C Bradshaw, P Flecknell and J Roughan

Direct comparison of vasectomized males and genetically sterile Gapdhs knockout males for the induction of pseudopregnancy in mice 365 W Garrels, D Wedekind, I Wittur, U Freischmidt, D Korthaus, T Ru¨licke and M Dorsch

Softened food reduces weight loss in the streptozotocin-induced male mouse model of diabetic nephropathy 373 SA Nørgaard, FW Sand, DB Sørensen, KSP Abelson and H Søndergaard

Partial cage division significantly reduces aggressive behavior in male laboratory mice 384 BR Tallent, LM Law, RK Rowe and J Lifshitz

A longitudinal study on timing and velocity of rat molar eruption: Timing of rat molar eruption 394 BJ Denes, A Lagou, D Dorotheou and S Kiliaridis

Creating effective biocontainment facilities and maintenance protocols for raising specific pathogen-free, severe combined immunodeficient (SCID) pigs 402 EJ Powell, S Charley, AN Boettcher, L Varley, J Brown, M Schroyen, MK Adur, S Dekkers, D Isaacson, M Sauer, J Cunnick, NM Ellinwood, JW Ross, JCM Dekkers and CK Tuggle

Short Report Brain perfusion fixation in male pigs using a safer closed system 413 GU Musigazi, S De Vleeschauwer, R Sciot, E Verbeken and B Depreitere

Case Report Malignant lymphoma with middle ear involvement in a Sprague-Dawley rat 418 J-F Lafond and A Landry

News Achievements of FELASA over the past 40 years 427

Workshop sobre Clasificacio´n de Severidad en Madrid 429 E Hevia

ECLAM/ESLAV Pre-Conference Workshop: Publishing Your Science: Toolkit and Guidelines 436

Calendar of events/Index to advertisers 440

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Abstract Scientists using laboratory animals are under increasing pressure to justify their sample sizes using a ‘‘power analysis’’. In this paper I review the three methods currently used to determine sample size: ‘‘tradition’’ or ‘‘common sense’’, the ‘‘resource equation’’ and the ‘‘power analysis’’. I explain how, using the ‘‘KISS’’ approach, scientists can make a provisional choice of sample size using any method, and then easily estimate the effect size likely to be detectable according to a power analysis. Should they want to be able to detect a smaller effect they can increase their provisional sample size and recalculate the effect size. This is simple, does not need any software and provides justification for the sample size in the terms used in a power analysis.

Keywords 3Rs, ethics and welfare, policy, reduction, sample size, statistics, techniques

Date received: 9 June 2017; accepted: 4 September 2017

Introduction ‘‘resource equation’’ and the ‘‘power analysis’’) and shows that they are related. A scientist can make a There is a crisis in pre-clinical biomedical research provisional choice of a sample size using ‘‘common involving laboratory animals. Too many papers publish sense’’ or the resource equation. Then, using the math- results which turn out to be irreproducible.1–3 One esti- ematics of the power analysis, he or she can easily mate puts the cost at $28 billion being wasted per check the magnitude of the response likely to be detect- annum in the United States alone.4 able for a specified level of power, significance, and The causes of this irreproducibility crisis have not sidedness. All that is needed is an estimate of the stand- been fully identified. But it has been known for many ard deviation (SD), Table 1 and some simple arith- years that experiments are often poorly designed, inad- metic. Scientists can easily investigate the effect of equately analysed, and misreported.5–7 A survey of changing the provisional sample size should they wish 271 papers chosen at random involving rats, mice to do so. and non-human primates8 showed that 87% did not report random allocation of experimental subjects to Current methods of determining the treatments and 86% did not report ‘‘blinding’’ sample size when measuring the results. None of the papers gave any justification for their choice of sample size, and a It is assumed that a proposed experiment has two substantial number of papers failed even to state the groups, namely ‘‘control’’ and ‘‘treated’’, and the sex, age or weight of the animals. Such failures can dependent variable is, or can be made, suitable for stat- lead to too many false-positive results.9 It has also istical analysis using a t-test or an analysis of variance. been suggested,10 on somewhat debatable evidence, that many animal experiments are under-powered, Medical Research Council Toxicology Unit, University of Leicester, leading to large numbers of false-negative results. If UK these remain unpublished, the proportion of published false-positive results due to the use of a 5% significance Corresponding author: Michael FW Festing, c/o MRC Toxicology Unit, Hodgkin Building, level will be increased. BO Box 138, Leicester, LE1 9HN, United Kingdom of Great Britain This note reviews the three methods of determining and Northern Ireland. sample size (‘‘tradition’’ or ‘‘common sense’’, the Email: [email protected] 342 Laboratory Animals 52(4)

Table 1. Cohen’s d (SESs) for sample sizes of 4–34 sub- experimental design11,12 and is the ﬁrst winner of the jects per group assuming 80% and 90% power, a 5% sig- ‘‘International Statistics Prize’’, so his views should be nificance level and a one-sided or two-sided test. taken seriously. Sample 80% 90% 80% 90% size one-sided one-sided two-sided two-sided 2. ‘‘The resource equation’’

4 2.00 2.35 2.38 2.77 This method13 is based on previous experience lar- 5 1.72 2.03 2.02 2.35 gely from agricultural and industrial research. The 6 1.54 1.82 1.80 2.08 equation is: 7 1.41 1.66 1.63 1.89 8 1.31 1.54 1.51 1.74 E ¼ ðÞthe total number of experimental units 9 1.23 1.44 1.41 1.63 ðÞthe number of treatment groups 10 1.16 1.36 1.32 1.53 E should be chosen to be between about 10 and 20, 11 1.10 1.29 1.26 1.45 although these are not rigid limits. 12 1.05 1.23 1.20 1.39 This method recognises that there is a slight ‘‘sweet 13 1.00 1.18 1.15 1.33 spot’’ within these two limits. If fewer animals were to 14 0.97 1.14 1.10 1.27 be used than the lower limit, then the chance of a type 15 0.93 1.10 1.06 1.23 II error (false-negative result) increases substantially. If 16 0.90 1.06 1.02 1.18 more animals were to be used than the upper limit, then 17 0.87 1.03 0.99 1.15 the cost and use of animals will increase for only a 18 0.85 1.00 0.96 1.11 modest gain. The method also shows that if there are 19 0.82 0.97 0.93 1.08 more than two treatments, the number of experimental 20 0.80 0.94 0.91 1.05 subjects per treatment can be reduced. The method is a 21 0.78 0.92 0.89 1.03 useful addition to ‘‘common sense’’. But funding organ- 22 0.76 0.90 0.86 1.00 isations and ethical review committees are increasingly demanding the use of a power analysis to determine 24 0.73 0.86 0.83 0.96 sample size. Apparently they are under the (false) 26 0.70 0.82 0.79 0.92 impression that it provides an objective method of 28 0.67 0.79 0.76 0.88 determining sample size. 30 0.65 0.76 0.74 0.85 32 0.63 0.74 0.71 0.82 3. ‘‘Power analysis’’ 34 0.61 0.72 0.69 0.80 Introduced by Jacob Cohen in in the 1960s,14 this method depends on a mathematical relationship The determination of sample size for discrete data is between six variables. If five of these are specified, the not discussed here. sixth one (usually sample size) can be estimated, using It is also assumed that the aim is to design an experi- dedicated software. ment which is both small and powerful. The six variables and some of the factors which influence them are shown in Figure 1. 1. ‘‘Tradition’’ or ‘‘common sense’’ 1. The SD Currently, most investigators choose sample sizes which were used, apparently successfully, by other An estimate of the SD of the character of interest investigators conducting similar work. Given the wide should be obtained from previous experiments invol- range of types of experiment, independent and depend- ving animals of the same species, strain, age, gender ent variables, experimental units, species, strains and and health status as the animals which are to be used. outcome variables in laboratory animal research, this If it is not available a pilot study using small numbers seems to be a sensible approach. Cox and Reid11 state of untreated animals of the same strain etc. will that ‘‘Except in rare instances..., a decision on the size be needed. of the experiment is bound to be largely a matter of Powerful small experiments require tight control of judgement and some of the more formal approaches to the inter-individual variation. This depends on several determining the size of the experiment have spurious factors, shown in Figure 1. The animals (or other precision’’. They are probably referring to the ‘‘power experimental subjects) should be as uniform as possible. analysis’’. Sir David Cox is the author of two books on Within-subject and randomised block designs15 are Festing 343

Experimental unit Experimental design (e.g. within/ between (completely randomised, randomised block, other)

Genec variaon (inbred/outbred) 5.Variability (SD) Environmental Availability variaon/infecon

1.Provisional Sample Standardised eﬀect 6 .Esmated minimum Character size(from previous size. SES detectable eﬀect size sensivity experience)

Dose level 2.Power (specify 80-90%?)

3.Signiﬁcance level (Specify 0.05?)

4.Sidedness (Specify)

Figure 1. The six factors directly used in a power analysis (labelled 1–6), and factors which may influence them. Usually a power analysis is used to determine sample size (6) for specified levels of the other five variables. However, here the aim is to determine the effect size (2), for a given sample size, for reasons which are explained in the text.

likely to give better control of variation than between- 3. The power subject designs.16 Variation will be minimised if the experimental animals are free of clinical or sub-clinical This is the probability that the experiment will infection and have been raised in a good environment. reject the null hypothesis when it is false. A power They should be of uniform age and weight. Genetic of 80% or 90% is usually specified. For any given variation can be controlled by using inbred or F1 sample size there is a complete range of levels of hybrid strains of mice and rats.17 power and an associated ES that the experiment is Measurement error needs to be minimised. likely to be able to detect. This is explained in more Duplicate or repeat determinations of the outcome detail below. variable can sometimes be used to reduce such vari- Note that if a completed experiment has rejected ation.18 The experimental data need to be of high qual- the null hypothesis it was clearly powerful (although ity and collected using ‘‘Good Laboratory Practice’’ it could be a false-positive result). However, the con- standards, and staff should be well trained in hus- verse is not true. If an experiment fails to reject the bandry and the collection of the data. null hypothesis it could be either because it lacked power or because there was no treatment effect of 2. The effect size (ES) sufficient size to be worth trying to detect. In a power analysis the aim is only to design experiments This is the difference between the means of the two to be able to detect ESs which are sufficiently large to groups which are being compared. Large ESs are easier be of scientific interest. to detect than small ones. So, when planning an experiment the aim should be to give as high a dose (or 4. The significance level equivalent) as possible, but not so high as to cause unwanted side effects. This is the probability that the experiment will pro- Where possible choose sensitive strains and spe- duce a false-positive result (a type I or a error). It is cies of animals, or avoid insensitive ones. For example, usually set at p ¼ 0.05. So in a well-designed and Sprague-Dawley rats from one commercial supplier are unbiased experiment there is a 5% chance of making not suitable for studies of the endocrine disruptor bis- a type I (false-positive) error. Occasionally a case can phenol A as they are insensitive to steroid substances.19 be made for using a different level. But specifying a 1% Some variables are more sensitive than others, so more significance level, for example, would increase the sensitive ones should be chosen where possible. required sample size or decrease the power. 344 Laboratory Animals 52(4)

5. The sidedness of the test animals are genetically identical. All these factors lead to lower SDs. Higher responses may also be obtained. A two-sided test, in which the mean of the treated Higher dose levels of test substances can be given and group could be either larger or smaller than the mean of sensitive species and strains can often be chosen or the control group, is usually used. But if the response insensitive ones avoided. can only go, or would only be of interest, in one direc- As a result, much higher SESs are observed in tion then a one-sided test should be used. A one-sided laboratory animal experiments than in clinical trials. test leads to a more powerful experiment or requires a Here it is suggested that SES of 1.1 ‘‘extra-large’’, 1.5 smaller sample size. ‘‘gigantic’’ and 2.0 SDs ‘‘awesome’’ are added to take account of laboratory animal experiments, including 6. The sample size in vitro studies using animal cells or extracts. Detecting SES of these magnitudes would require This is the number of experimental subjects in each sample sizes of 17, 8, and 5 subjects per group, respect- group. Usually, a power analysis is used to estimate a ively, with an 80% power, a 5% significance level and a suitable sample size for a proposed experiment. two-sided t-test. However, the alternative, used here, is for the investi- Figure 2 (a)–(d) shows the estimated SESs from an gator first to choose a sample size based on ‘‘common experiment on the effect of chloramphenicol on four sense’’, previous studies and/or the ‘‘resource equation’’ haematological outcomes in mice of four inbred strains and then calculate the ES likely to be detectable using and one outbred stock at six dose levels. The raw data the mathematics of a power analysis, as incorporated in for these figures is included in the original publica- Table 1. This is explained below. This approach is easy tion.22 There are clear dose-related differences in to understand and is less prone to error than the more response. Many SESs are ‘‘gigantic’’, or ‘‘awesome’’, conventional approach. being well over two SDs. There are clear strain differences in sensitivity. For example, the outbred CD-1 The ‘‘standardised effect size’’ stock was relatively more resistant to chloramphenicol (SES or Cohen’s d) for all four characters than the four inbred strains, and the white blood cell count (WBC) response to The SES or Cohen’s d is a useful statistic. It is the ES chloramphenicol in strain C3H was much higher than divided by the pooled SD (SDpooled). So it is the mag- in other strains. nitude of the difference between the means of two It is not necessary to know how to calculate the SESs groups in units of SDs. The SES is widely used when when using them in estimating sample size as discussed combing the results of several studies in a meta-analy- below. But investigators are encouraged to quote the sis.20 It can also be used when comparing the treatment observed SESs from their completed experiments. response for different variables because they are all Details are given in the Appendix. expressed in the same units (SDs). In toxicity tests, for example, measurements of haematology, clinical The relationship between the SESs and biochemistry, organ weights and other factors can be sample size. combined to give an over-all response to a test chemical in SD units.21 Moreover, the SES is directly related to Figure 3 shows the relationship between sample sample size if the power, sidedness and significance level size and SESs, over the range of 4–34 animals in a proposed experiment are fixed. (or other experimental units) per group for a signifi- Based on human studies Cohen (who was a cance level of 0.05 and a two-sided test, for power psychologist) suggested that responses to a treatment levels of 60%–90%. resulting in SESs of 0.2, 0.5 and 0.8 SDs would repre- Note that for any given sample size there is a range sent small, moderate and large treatment responses of SESs and power levels likely to be detectable. For requiring sample sizes of 394, 64, or 26 subjects per example, with six animals per group there will be a group, respectively, to detect the effect. This is assum- 90% chance of detecting an SES of about 2.1 SDs, an ing an 80% power, a 5% significance level and a two- 80% chance of detecting an SES of 1.8 SDs, a 70% sided t-test. chance of detecting an SES of 1.6 SDs and a 60% However, laboratory animals are intrinsically much chance of detecting an SES of 1.4 SDs and so on more uniform than humans, so the SDs are lower. down to a 5% chance of detecting a non-existent Groups of animals can be obtained of similar age and response (a type I error). As a consequence, an investi- weight, free of clinical or sub-clinical infection, fed the gator will sometimes be ‘‘lucky’’ and detect an effect same diet and housed in the same environment. Inbred which is smaller than the experiment was designed to strains of mice and rats can also be used in which all be able to detect. Anyone wanting to repeat an Festing 345

Red blood cells White blood cells

C3H C3H

BALB/c C57BL C57BL CBA CBA CD−1 CD−1 BALB/c SES, Standard deviations SES, SES, Standard deviations SES, −1012345 −1012345 500 1500 2500 500 1500 2500 Dose Dose Reticulocytes Haemoglobin

CBA C3H C3H C57BL BALB/c BALB/c C57BL CD−1 CBA CD−1 02468 SES, Standard deviations SES, Standard deviations SES, −1012345 500 1500 2500 500 1500 2500 Dose Dose

Figure 2. Observed standardised effect sizes (SES) for four haematological parameters in mice treated with chloramphenicol at six dose levels (mg/kg). Note important strain and dose level effects. experiment should use a larger sample size than was used The KISS approach combines these two methods. in the original experiment. Scientists make a provisional estimate of the sample Table 1 gives the corresponding SES for sample sizes size using ‘‘common sense’’ and/or the resource equa- ranging from 4–34 subjects per group for 80% and 90% tion, then use a table and some simple arithmetic to power and a 5% signiﬁcance level, one sided or two estimate the ES that the experiment is likely to be sided. A more extensive table is given by Ellis.20 able to detect for a given power, etc. Optionally, they may express this ES as a percentage change. If, on The ‘‘Keep It Simple, Stupid’’ (KISS) reﬂection, they want to be able to detect a smaller ES approach to the determination they can increase the provisional sample size and re-do of sample size the calculations. They can then legitimately explain their choice in terms of the power analysis. Most investigators base sample sizes on past experi- The procedure is as follows: ments which appear to have given satisfactory results. Given the wide range of variables shown in 1. Plan the experiment. Figure 1, this makes sense.11 However, funding organisations and ethical review committees often require Specify the purpose of the experiment and consider scientists to justify their sample sizes using a power whether comparable results could be obtained from analysis. using methods which do not involve live animals. 346 Laboratory Animals 52(4) Standardised effect size Standardised effect 90% 80% 70% 60% 0.6 1.0 1.4 1.8 2.2 2.6 3.0

4 5 6 7 8 9 10 12 14 16 18 Sample size per group

Figure 3. Standardised effect size (SES or Cohen’s d) as a function of sample size (per group) for four levels of power (60%–90%) assuming a two-sided t-test with a 5% significance level and a quantitative dependent variable. The vertical dotted lines show the range of sample sizes using the ‘‘resource equation’’ method of determining sample size

Assuming that the use of animals is essential, specify percentage change if it would make it easier to the species, strain, age/weight and gender of the ani- understand. mals to be used. Identify the ‘‘experimental unit’’ (this 5. Decide whether the ‘‘predicted detectable ES’’ is is the unit of randomisation and of statistical analysis. acceptable (i.e. whether it will detect a sufficiently Any two experimental units must be able to receive small effect, should it be present). If not then different treatments). Specify treatments (doses, meth- choose a larger provisional sample size and re-do ods of administration), number of treatment groups, the calculations. outcome variables to be measured, timeline, and experi- 6. In the Materials and methods section of the result- mental design (e.g. completely randomised, randomised ing publication, and in accordance with the Animal block, factorial, other). Research: Reporting of In Vivo Experiments (ARRIVE) guidelines,23 a statement such as the 2. From previous studies, obtain one or more esti- following could be included: mates of the mean and SD of the variable of interest in control subjects. It may be best to choose a ‘‘A power analysis shows that the sample size of high and a low SD. XX has a XX% power to detect an effect size of 3. Choose a provisional sample size based on previous XX (units or %) assuming a 5% significance level studies, the literature, the resource equation and and an XX-sided test.’’ ‘‘common sense’’. 4. Find the SES for the provisional sample size Where the XXs are replaced by the appropriate values. in Table 1, with the desired power level and sidedness of the test. Multiply the SES by the SD to In order to avoid publication bias, the results of the give the ‘‘predicted detectable ES’’ for the chosen experiment should be written up and submitted for levels of power, etc. This can be expressed as a publication whether or not the observed differences Festing 347

Table 2. Estimated detectable effect size (ES) and % change in some haematological and clinical biochemistry characters in outbred Sprague-Dawley rats assuming two treatment groups (‘‘Treated’’ and ‘‘Control’’) for sample size N ¼ 12 (SES ¼ 1.39, Table 1).

Estimated detectable Estimated Biomarker Units Sex Mean SD ESa in SDs % change

RBC (106/ll) Male 8.61 0.27 0.38 4 HGB (g/dl) Male 15.80 0.40 0.56 4 WBC (103/ll) Male 9.71 2.50 3.48 36 RBC (106/ll) Female 8.29 0.32 0.44 5 HGB (g/dl) Female 15.70 0.60 0.83 5 WBC (103/ll) Female 5.69 1.48 2.06 36 AST (U/l) Male 105.00 23.00 31.97 31 ALT (U/l) Male 36.00 8.00 11.12 31 SDH (U/l) Male 7.30 4.60 6.39 88 AST (U/l) Female 117.00 33.00 45.87 39 ALT (U/l) Female 42.00 18.00 25.02 60 SDH (U/l) Female 13.20 5.50 7.65 58 aThis assumes a 90% power a 5% significance level and a two-sided test. It is the SD SES (1.39 for a sample size of 12 in Table 1). RBC: red blood cell count; HGB: haemoglobin; WBC: white blood cell count; AST: aspartate aminotransferase; ALT: alanine aminotransferase; SDH: sorbitol dehydrogenase; SES: standardised effect size.

were ‘‘statistically significant’’. Negative results are of Example 2 extra value when backed up by a power analysis as shown because they help to preclude a large undetected An investigator plans to study the effect of a drug on effect. chosen haematological and biochemical characters in Sprague-Dawley rats. Means and SDs are taken from 25 Example 1 a published paper. A sample size of 12 rats per group is proposed. Question: ‘‘Does a potential new drug alter red blood The calculations are shown in Table 2. Quite small cell (RBC) count in mice?’’ changes of the order of 4% to 5% in RBC and haemo- From a published study, C57BL/6 female mice had a globin (HGB) are likely to be detectable, but only large mean RBC count of 9.19 with an SD of 0.70 (n/ml). changes will be detectable in WBCs and clinical (Make sure that it is the SD not the SEM.) biochemistry. Note that the power analysis does not Suppose a provisional sample size of n ¼ 12 mice/ predict the actual magnitude of the response, only group is chosen, based on previous studies. how large the response would need to be to be detect- From Table 1 for a sample size of 12 with a 90% able. So, for example, if the ES as a percentage of the power and a two-sided test, SES ¼ 1.39. mean for WBC is 36% or greater, then it will probably Therefore the ‘‘predicted detectable ES’’ (SES*SD) is be detected assuming the SDs are about the same as 1.39 0.70 ¼ 0.97 (n/ml). those published in the original paper. Or as a percentage ¼ (0.97/9.19) 100 ¼ 11%. Having performed the calculations, the investiga- Assuming that this ‘‘predicted detectable ES’’ is tor has still to decide whether the sample size is judged to be acceptable, a sample size of 12 mice per appropriate. group can be used. 24 Following the ARRIVE guidelines, a statement More than two groups such as the following should be written in the Materials and methods section The KISS method estimates the ES that a comparison between any two groups is likely to be able to detect for ‘‘A power analysis shows that the sample size of 12 the specified sample size, power, significance level and mice/group has a 90% power to detect an ES of 0.97 sidedness of the test. If another group (say an inter- n/ml or an 11% change, assuming a 5% significance mediate dose or a qualitatively different treatment) of level and a two-sided test.’’ the same size is added then the same calculations apply 348 Laboratory Animals 52(4) to it. However, with more than two groups there will References be a better estimate of the SD so sample size can be 1. Begley CG and Ellis LM. Drug development: Raise slightly reduced. The resource equation method should standards for preclinical cancer research. Nature 2012; give some guidance on this, but the dangers of ‘‘spuri- 483: 531–533. ous precision’’ and the importance of ‘‘common sense’’ 2. Scott S, Kranz JE, Cole J, et al. Design, power, and inter- should not be forgotten. pretation of studies in the standard murine model of If an additional factor such as gender is added in a ALS. Amyotroph Lateral Scler 2008; 9: 4–15. factorial design so that there are four groups (male and 3. Prinz F, Schlange T and Asadullah K. Believe it or not: female control and treated), then sample size is the How much can we rely on published data on potential number of males plus females in the treated and control drug targets? Nat Rev Drug Discov 2011; 10: 712. 4. Freedman LP, Cockburn IM and Simcoe TS. The group. Economics of reproducibility in preclinical research. Formal power analysis is available for experiments PLoS Biol 2015; 13: e1002165. with several treatment groups, but it is subject to even 5. Festing MF. We should be designing better experiments. more ‘‘spurious precision’’ than if just two groups are Vet Anaesth Analg 2003; 30: 59–61. involved. 6. Festing MF. Principles: The need for better experimental design. Trends Pharmacol Sci 2003; 24: 341–345. Discussion 7. Festing MFW. The scope for improving the design of laboratory animal experiments. Lab Anim 1992; 26: No one method of determining sample size is entirely 256–267. satisfactory. ‘‘Common sense’’ may work well with an 8. Kilkenny C, Parsons N, Kadyszewski E, et al. Survey of experienced investigator who is thoroughly familiar with the quality of experimental design, statistical analysis and his or her material and has already performed a number reporting of research using animals. PLoS One 2009; 4: of experiments similar to the one proposed. But it is less e7824. 9. Bebarta V, Luyten D and Heard K. Emergency medicine satisfactory for those starting a new research topic. The animal research: Does use of randomization and blinding resource equation method provides a useful rule of affect the results? Acad Emerg Med 2003; 10: 684–687. thumb method for avoiding experiments which are 10. Button KS, Ioannidis JP, Mokrysz C, et al. Power failure: probably either too small, so likely to lead to false-nega- Why small sample size undermines the reliability of tive results, or unnecessarily large leading to a waste of neuroscience. Nat Rev Neurosci 2013; 14: 365–376. resources. But it doesn’t have the (possibly spurious) 11. Cox DR and Reid N. The theory of the design of experi- mathematical justification of the power analysis. ments. Boca Raton, FL: Chapman and Hall/CRC Press, The power analysis is complex and it involves a sub- 2000. jective element because the investigator must decide the 12. Cox DR. Planning experiments. NY: John Wiley and minimum ES likely to be of scientific interest. It also suf- Sons, 1958. fers from spurious precision because there are several 13. Mead R. The design of experiments. Cambridge, NY: important variables, such as the sensitivity of the Cambridge University Press, 1988. 14. Cohen J. Statistical power analysis for the behavioral sci- chosen experimental material, which are not taken into ences. Hillsdale NJ: Lawrence Erlbaum Associates, 1988. account. Normally, it also requires access to specialised 15. Festing MF. Randomized block experimental designs can software which, although readily available, requires an increase the power and reproducibility of laboratory additional level of understanding. If scientists are animal experiments. ILAR J 2014; 55: 472–476. required to use unfamiliar software and unfamiliar vari- 16. Festing M, Overend P, Cortina Borga M, et al. The design ables, there is a danger that their calculations will be of animal experiments, 2nd ed. London, UK: Sage incorrect. The KISS approach of choosing sample size Publications, 2016. using ‘‘common sense’’, and/ or the resource equation 17. Festing MF. Genetically defined strains in drug develop- and combining it with the power analysis provides a sim- ment and toxicity testing. Methods Mol Biol 2016; 1438: plified solution to the problem of determining sample size 1–17. in laboratory animal experiments. 18. Karp NA, Baker LA, Gerdin AK, et al. Optimising experimental design for high-throughput phe- Declaration of Conflicting Interests notyping in mice: A case study. Mamm Genome 2010; 21: 467–476. The author(s) declared no potential conflicts of interest with 19. Richter CA, Birnbaum LS, Farabollini F, et al. In vivo respect to the research, authorship, and/or publication of this effects of bisphenol A in laboratory rodent studies. article. Reprod Toxicol 2007; 24: 199–224. 20. Ellis PD. The essential guide to effect sizes. Cambridge: Funding Cambridge University Press, 2010. The author(s) received no financial support for the research, 21. Festing MF. Extending the statistical analysis and authorship, and/or publication of this article. graphical presentation of toxicity test results using Festing 349

2 2 standardized effect sizes. Toxicol Pathol 2014; 42: SDp ¼ Sqrt ((S 1 þ S 2)/2), where the ‘‘Ss’’ are the SD 1238–1249. for the two groups, respectively. 22. Festing MF, Diamanti P and Turton JA. Strain differ- If the group sizes are unequal, then a weighted mean ences in haematological response to chloramphenicol suc- 2 2 is used. Pooled SDp ¼ Sqrt ((n1–1) S 1 þ (n2–1) S 2)/ cinate in mice: Implications for toxicological research. {(n1–1) þ (n2–1)}. Food Chem Toxicol 2001; 39: 375–383. The SES can also be calculated as t/sqrt (N), where 23. Kilkenny C and Altman DG. Improving bioscience research reporting: ARRIVE-ing at a solution. Lab ‘‘t’’ is Student’s t. Anim 2010; 44: 377–378. These SESs are biased estimates of the population 24. Kilkenny C, Browne WJ, Cuthill IC, et al. Improving SES. To correct for this, the SESs is multiplied by the bioscience research reporting: The ARRIVE guidelines factor shown below. However, this can be ignored if the for reporting animal research. PLoS Biol 2010; 8: pooled sample sizes are greater than 10. e1000412. ÈÉ 25. Delaney B, Karaman S, Roper J, et al. Thirteen SESunb ¼ 1 ½3=ðÞ4df 1 SES week rodent feeding study with grain from molecular stacked trait lepidopteran and coleopteran protected where ‘‘df’’ is the number of degrees of freedom in a (DP-ØØ4114–3) maize. Food Chem Toxicol 2013; 53: t-test using this number of animals. 417–427. Table A1 shows the above adjustment factors for 26. Perron IJ, Pack AI and Veasey S. Diet/Energy balance affect sleep and wakefulness independent of body weight. multiplying with the estimates of the SES. Sleep 2015; 38: 1893–1903. Example Effect of diet accessibility on sleep in mice26. Mice Appendix and rats sleep in short bouts throughout the 24-hour Calculating SESs following an experiment period. In a study of diet and energy balance in It is not necessary to know how to calculate the SESs in order to use them to assess sample size as outlined Table A1. Correction to obtain an above. However, investigators are encouraged to quote unbiased estimate of a standar- the SESs which they have found in their experiments. dised effect size (see text for It would certainly help in designing future experiments details). and in showing that, with proper control of the varia- Degree of tion, ‘‘extra-large’’, ‘‘gigantic’’ and ‘‘awesome’’ SES can freedom Multiplier sometimes be observed. The SES is ES/SDp, where SDp is the pooled SD 3 0.73 and the ES is the diﬀerence between the means of the 4 0.80 two groups being compared. 5 0.84 If an analysis of variance has been used to analyse 6 0.87 the results, then the SDp is the square root of the error 7 0.89 mean square in the analysis of variance table. 8 0.90 Alternatively, if only means and observe SDs are avail- 9 0.91 able, and group sizes are equal it can be estimated as 10 0.92 the square root of the mean of the two variances:

Table A2. Estimation of the observed standardised effect size (SES) from the results of an experiment on bouts of sleep in mice (Perron et al.26). The SES is the difference in means divided by the pooled SD.

SD (SEM Variance Pooled Treatment Mean SEM N/group Sqrt (N)) (SD2) SD

Control 297.5 14.5 13 52.28 2733.25 Treated 408.4 19.3 13 69.59 4842.37 110.9a 3787.81b 61.55 SES 110.9/61.55 ¼ 1.80

aDifference between means. bMean of the variances 350 Laboratory Animals 52(4)

C57BL/6 mice, the control mice had an average of obtain the observed SES following the experiment are 297.5 14.5 (mean SEM, N ¼ 13) bouts of sleep per shown in Table A2. The SEMs need to be converted to 24 hours, whereas the treated mice had 408 19.3 bouts SDs then to variances, then averaged and the square (mean SEM, N ¼ 13). roots calculated. The observed SES was a ‘‘gigantic’’1.8 That represents a change of 37%, and it was found SDs. No correction for bias is necessary with these to be statistically highly signiﬁcant. The calculations to sample sizes.

Re´sume´ Il est de plus en plus instamment demande´ aux scientifiques utilisant des animaux de laboratoire de justifier la taille de leur ećhantillon a` l’aide d’une « analyse de puissance ». Dans cet article, je passe en revue les trois me´thodes actuellement utiliseés pour de´terminer la taille de l’ećhantillon : « Tradition » ou « bon sens », « l’e´quation des ressources » et « l’analyse de puissance ». J’explique comment, en utilisant l’approche « KISS », un scientifique peut choisir de manie`re provisoire la taille de l’ećhantillon a` l’aide de n’importe quelle me´thode, puis facilement estimer la taille susceptible d’eˆtre de´tectable selon une analyse de puissance. S’ils veulent eˆtre en mesure de de´tecter un effet moindre ils peuvent augmenter la taille de leur ećhantillon provisoire et recalculer la taille susceptible de produire un effet. Cette me´thode simple, sans qu’aucun logiciel ne soit nećessaire, permet de justifier la taille de l’ećhantillon selon les termes utilise´s dans une analyse de puissance.

Abstract Wissenschaftler, die Labortiere verwenden, stehen unter zunehmendem Druck, ihre Versuchsgro¨ße mittels einer ÐPoweranalyse‘‘ zu rechtfertigen. In diesem Artikel bespreche ich drei Methoden, die derzeit zur Bestimmung von Stichprobengro¨ßen dienen: ÐTradition‘‘ oder Ðgesunder Menschenverstand‘‘, die ÐRessourcengleichung‘‘ und die ÐPoweranalyse‘‘. Ich erlaütere, wie Wissenschaftler auf Basis des ÐKISS‘‘- Konzepts eine vorlaüfige Wahl der Stichprobengro¨ße mittels einer beliebigen Methode treffen und anschließend einfach die voraussichtlich nachweisbare Behandlungseffektgro¨ße gema¨ß einer Poweranalyse scha¨tzen ko¨nnen. Wenn sie eine geringere Effektgro¨ße aufdecken wollen, ko¨nnen sie ihre vorlaüfige Probengro¨ße erho¨hen und die Effektgro¨ße neu berechnen. Dies ist einfach, kann ohne Einsatz von Software erfolgen und liefert die Rechtfertigung fu¨r die Stichprobengro¨ße gema¨ß den bei einer Poweranalyse zur Anwendung kommenden Werten.

Resumen Los cientı´ficos que utilizan animales de laboratorio estań sometidos cada vez a ma´s presioń para justificar sus tamanõs de muestra utilizando un ‘‘ana´lisis de poder". En este estudio se analizan los tres me´todos utilizados actualmente para determinar el tamanõ de las muestras: ‘‘Tradicioń’’ o ‘‘Sentido comuń", la ‘‘Ecuaciońde recursos’’ y el ‘‘Ana´lisis de poder". Explico co´mo utilizando el me´todo ‘‘KISS’’ los cientı´ficos pueden tomar una decisioń profesional sobre el tamanõ de las muestras a trave´s de cualquier me´todo, y luego estimar faćil- mente el tamanõ en concreto ma´s detectable seguń un ana´lisis de poder. Si desean detectar un efecto inferior pueden incrementar su tamanõ de muestra provisional y recalcular el tamanõ del efecto. Esto es simple, no se requiere ninguń software y justifica el tamanõ de la muestra en los te´rminos utilizados en un ana´lisis de poder. laboratory an imals Original Article limited Laboratory Animals 2018, Vol. 52(4) 351–364 ! The Author(s) 2017 Analgesics promote welfare and sustain Reprints and permissions: sagepub.co.uk/journalsPermissions. tumour growth in orthotopic 4T1 and B16 nav DOI: 10.1177/0023677217739934 mouse cancer models journals.sagepub.com/home/lan

Jennifer Lofgren1, Amy L Miller2, Claudia Chui Shan Lee3, Carla Bradshaw3, Paul Flecknell3 and Johnny Roughan3

Abstract Murine orthotopic cancer models often require surgery, potentially causing pain or distress. However, analgesics are often withheld because they may alter tumour development. Two orthotopically implanted cancers were investigated in mice pre-treated with meloxicam (10 mg/kg), buprenorphine (0.2 mg/kg) or saline (1 ml/kg). Tumours were imaged and welfare was assessed using body weight, behaviour and nociceptive responses. In study 1, BALB/c mice were inoculated with 4T1 mammary carcinoma or saline during surgery or anaesthesia. As pre-treatment with a single buprenorphine dose appeared beneficial to cancer growth consistency, a second cohort of mice additionally received saline or buprenorphine at 12 and 24 h. Surgery resulted in increased mammary tumour growth and lung metastases. These unwanted effects were lessened by buprenorphine pre-treatment, especially when given repeatedly. Mammary tumour-bearing mice became less active and nociceptive thresholds declined over time, indicating some discomfort as tumours grew. In study 2, C57BL/6 mice received B16 melanoma. This non-surgical model was used to determine whether meloxicam or buprenorphine affected cancer seeding of the lungs. While meloxicam reduced B16 lung seeding, buprenorphine did not. Mechanical thresholds decreased as cancer developed in mice bearing melanoma, but the magnitude of this was insufficient to conclude that there were any significant welfare concerns. This study highlights the scientific value in utilising non-surgical models, where possible. When surgery must be performed at the time of tumour inoculation, the effects of this should be controlled with appropriate analgesics to enhance the value and possibly translation of the research.

Keywords mouse, buprenorphine, NSAID, meloxicam, analgesia, cancer, refinement

Date received: 14 December 2016; accepted: 13 September 2017

Large numbers of mice are involved in cancer research. analgesics are often withheld due to concerns about Many receive orthotopic tumour inoculation, whereby their potentially confounding effects on tumour devel- tumours grow in the tissue of origin. This approach is opment. A search for the terms ‘mouse AND tumour supposed to maximise the translational relevance of 1,2 results, whereas with heterotopic tumour inoculation 1Unit for Laboratory Animal Medicine, University of Michigan tumours grow in an unrelated tissue, for example sub- Medical School, Ann Arbor, Michigan, USA 2 cutaneously. The tumour cells implanted may be from School of Agriculture, Food and Rural Development, Newcastle University, Newcastle upon Tyne, UK different strains of mice or even different species. They 3Pain and Animal Welfare Science Group, Institute of can be grown in immunocompromised mice, or as in Neuroscience, Comparative Biology Centre, Newcastle this case, they can be syngeneic derived tumour cell University, Newcastle upon Tyne, UK lines. Although tumour inoculation can be a minimally invasive procedure, some procedures such as intra- Corresponding author: Johnny Roughan, Newcastle University, The Medical School, ocular injection may not be, and some models require Framlington Place, Newcastle upon Tyne, NE2 4HH, United surgery, for example laparotomy to implant hepatocel- Kingdom of Great Britain and Northern Ireland. lular carcinoma. Although this could be painful, Email: [email protected] 352 Laboratory Animals 52(4)

AND surgery’ on https://www.ncbi.nlm.nih.gov As concerns about using analgesics in cancer studies resulted in 49,266 articles. When combined with the mainly relate to tumour growth alterations, the inten- terms ‘analgesia’, ‘opioid OR buprenorphine’, or tion was to investigate from a ‘severe case’ perspective, ‘nonsteroidal anti-inflammatory drug (NSAID) OR so the analgesic dose rates were purposefully high com- NSAID OR meloxicam OR carprofen OR ketoprofen pared with those usually recommended.18 OR indomethacin OR acetaminophen OR paraceta- Welfare was assessed using body weight changes, mol’, that number decreased to 818 articles: a difference peripheral nociceptive sensitivity alterations (using of 48,448 articles concerning studies with both tumours von Frey and Hargreaves testing) and by recording and surgery in mice that listed no analgesics. It may be behaviour changes post-procedurally and as cancer that many of these actually used analgesia but did not developed. All mice were initially pre-treated with a disclose it.3 However, such under-reporting is not usu- single dose of meloxicam, buprenorphine or saline ally the case.4 Of those studies that did list analgesics, prior to 4T1 or B16 cancer inoculation. The 4T1 mam- most evaluated their effects on tumour-associated pain mary carcinoma cell line was tested as cells are inocu- or therapeutic effects on tumour growth. For example, lated orthotopically; however, this can be achieved whereas NSAIDS have been found to impede tumour either non-surgically or surgically, via percutaneous development in some orthotopic tumour models in injection, or following surgical exposure of the fat rodents,5–7 some opioids can enhance cancer growth, pad.19 We wished to assess whether, as has previously often via suppression of the immune system.8,9 been suggested,19 unnatural surgical implantation actu- However, pain or stress due to surgery, anaesthesia or ally provides a more reliable model of human breast even sub-optimal housing can also alter tumourigen- cancer, and evaluate the impact of distress or post- esis.10–16 Analgesics including buprenorphine and indo- surgical pain on tumour development. Study 1 there- methacin can minimise surgery-associated changes, fore had tumours implanted both in conjunction with bringing tumour development closer to that observed surgery or using anaesthesia only. Mice were inoculated in a non-surgical model. The mechanism could involve with 4T1 mammary carcinoma or saline, the latter to aspects such as preventing hypothalamic–pituitary– permit assessment of the effects of surgery or anaesthe- adrenal (HPA) axis up-regulation or minimising sia in the absence of analgesics or cancer. Having immunological impacts, such as maintaining natural assessed the effects of a single buprenorphine dose, killer cell functioning.8,17 study 1 was extended to examine multiple doses. The present investigations aimed to evaluate the Accordingly, a second cohort of mice was tested, with impacts of meloxicam or buprenorphine on tumour additional buprenorphine or saline being given at 12 growth in two orthotopic mouse cancer models: and 24 h following tumour inoculation. Initial analyses in BALB/c mice inoculated with 4T1 mammary car- found no indication that it would be beneficial to test cinoma, and then in C57BL/6NCrl mice with B16 multiple doses of meloxicam. melanoma. The 4T1 work also assessed the effects of The two cohorts in study 1 are respectively referred using surgery for tumour implantation, which rather to as the 4T1 single-dose and multiple-dose groups. All unusually is optional, and of using single- or multiple- mice in cohort 2 received 4T1 cancer inoculation. buprenorphine dosing. It was hypothesised that provi- Studies of B16 melanoma do not usually involve sur- sion of pain relief, rather than being confounding, gery. Cells are injected intravenously and primarily seed might both improve welfare and enhance study validity. the lungs. Therefore, in study 2 all mice were inoculated intravenously with B16 melanoma following a single pre-treatment with saline, meloxicam or buprenor- Materials and methods phine. The aim was to determine whether meloxicam Ethical approval or buprenorphine altered metastatic tumour spread to the lungs. All work was undertaken according to the Animals Group sizes were established using data from a pre- (Scientific Procedures) Act 1986 under Home Office vious study where between 8 and 10 mice per group Licence Authority with approval from the Newcastle achieved 80 to 85% power.20 University Animal Welfare Ethical Review Body (AWERB). Animals and husbandry Design Mice were supplied by Charles River (Margate, Kent, UK). They were certified free of the common pathogens Figure 1 shows the final mouse numbers and treat- listed on their website (http://www.criver.com/pro- ments. Individuals were assigned using a random ducts-services/basic-research/health-reports/europe- number generator (https://www.random.org). asia/uk-by-species). They weighed 18 0.25 g. For Lofgren et al. 353

Figure 1. Treatments combinations and final numbers. Mice were female BALB/c inoculated with saline or 4T1 mammary carcinoma (Study 1), or female C57BL/6NCrl inoculated with B16 melanoma (Study 2). 4T1 mice underwent laparotomy or only anaesthesia (Surgery ¼ Yes/No). Cohort 1 received one (Dose(f) ¼ Single) s/c pre-inoculation injection (Pre-Treat) of saline (1 ml/kg), meloxicam (Melox: 10 mg/kg) or buprenorphine (Bup: 0.2 mg/kg). Cohort 2 received the same saline or buprenorphine pre-treatment as cohort 1, but treatment was repeated at 12 and 24 h (Dose(f) ¼ Multiple). Boxes in the ‘Cancer’ column contain mouse numbers. Black boxes show groups inoculated with cancer and clear boxes those that were non-cancer controls. Mice in the B16 study (Study 2) did not have surgery (surgery ¼ N/A). All were inoculated with B16 melanoma after one s/c pre-treatment of either saline (1 ml/kg), meloxicam (10 mg/kg) or buprenorphine (0.2 mg/kg). study 1 (4T1 mammary carcinoma) 108 female BALB/c acclimatised for one week in groups of 2–3 in individu- mice were used. Study 2 used 30 female C57BL/6NCrl ally ventilated cages (Arrowmight, Hereford, UK) con- mice inoculated with B16 melanoma. These strains were taining hardwood bedding (Aspen, BS and S Ltd, chosen because they are syngeneic for the tumour cell Edinburgh, UK), a cardboard tube, chew blocks and line each was to be inoculated with. The 4T1 cells used sizzle nest (B and K Universal, Hull, UK). Food (R&M in study 1 were derived from spontaneous mammary no.3, SDS Ltd, Essex, UK) and tap water was provided tumours in BALB/c mice, whereas the B16 melanoma ad libitum. Cages were cleaned once per week. The cells used in study 2 were from C57BL/6 mice with holding room was kept at 22 2C and 27–40% humid- spontaneously developing melanoma. They were ity on a 0700–1900 light cycle. 354 Laboratory Animals 52(4) Tumour inoculation and treatments by treatment-blinded staff. All staff involved in data The tumour cells were obtained from Caliper Life collecting data were cross-trained to ensure consistency. Sciences (Waltham, MA, USA) and were cultured following vendor recommendations. They were confirmed Nociceptive testing pathogen free by the IMPACT Profile I (PCR) at the University of Missouri Research Animal Diagnostic Mechanical response thresholds were obtained using and Investigative Laboratory. The cells were lucifer- ‘The Mousemet’ electronic von Frey (eVF) device ase-expressing 4T1-luc2 mammary carcinoma (4T1) (Topcat Metrology, Ely, UK). Animals were placed (developed by Kim et al.21) or B16-F10-luc-G5 melan- into one of four separate raised runs that had oma (B16) (Xenogen Corporation, Alameda, Plexiglas sides and steel rod floors. After a 10 min California, USA). All inoculations were performed acclimation period six readings were obtained from between 8 and 11 a.m. in batches of 8–10 mice. each hind-paw, allowing at least 2 min between each. In the 4T1 study mice were anesthetised with isoflur- The probe force rise rate was 1 g/s applied when ani- ane in oxygen (4–5%; 0.5–1 l/min) and maintained at 2– mals were stationary. The 4T1 eVF thresholds were 3% isoflurane in oxygen (0.25–0.5 l/min) via a nose tested at baseline (between 12 and 4 p.m. the day cone. Body temperature was kept between 36C and before inoculation), at 24 h following recovery from 38C using a heat pad (Harvard Apparatus, anaesthesia, and then on days 7, 14, 19 and 22. Edenbridge, Kent, UK). Absence of pedal withdrawal In the B16 study eVF readings were taken at baseline, responses was used to ensure adequate depth. After eye 1 h and on days 5, 9, 13 and 17. Equipment malfunc- ointment was applied (Pliva Pharma Ltd., Zagreb, HR) tion meant baseline eVF readings were missed in the the mice were subcutaneously (s/c) injected with saline first 10 4T1 mice. (1 ml/kg), buprenorphine (0.2 mg/kg; ‘Vetergesic’, Thermal nociceptive testing used a Hargreaves Reckitt-Coleman, Hull, UK) or meloxicam (10 mg/kg; apparatus (Model 37370; Ugo Basile, Italy). This had Boehringer Ingelheim, Labiana Life Sciences S.A., six clear plastiglas enclosures (11 cm 17 cm 14 cm) Terrassa, Spain). The fourth mammary gland was to which mice were first acclimated for 5 min. then inoculated percutaneously with 100 ml Dulbecco’s Baseline latencies were obtained between 12 and Phosphate Buffered Saline (DPBS) containing 1 106 4 p.m. the day before tumour inoculation. In the 4T1 4T1-luc2 cells, or the same volume of 0.9% sterile study Hargreaves data were subsequently recorded at saline. 24 h, and on post-inoculation days 5, 8, 14, 20 and 22. Mice in the multiple-dose 4T1 study (cohort 2) were The B16 recordings were at baseline, 1 h and on days 5, subcutaneously treated with saline (1 ml/kg) or bupre- 9, 13 and 17. Heat intensity was 280 mW/cm2 with a norphine (0.2 mg/kg) before tumour inoculation, and 30 s cut-off time. Three readings were obtained from then at 12 and 24 h after tumour inoculation. All mice each hind-paw, allowing 2 minutes between each, in the 4T1 study were prepared as if for surgery; the again when mice were stationary. abdomen was first shaved and sprayed with chlorhexidine (Hydrex derma-spray, Leeds, UK), but only half Behaviour recordings subsequently underwent a 1.5 cm midline laparotomy lasting 15 min. This was completed exactly as previ- Mice were filmed individually for 10 min in three clear ously described,22 but without abrasion of the ileum, cages containing only sawdust (Type 1144B, and the skin was closed using a continuous subcuticular Techniplast UK Ltd, Northamptonshire, UK). suture (4/0 polydioxanone; Ethicon, Livingston, UK) Recordings were made between 1 and 4 p.m. using rather than mattress sutures. The same veterinary sur- three Canon Legria HFM 506 cameras placed 30 cm geon performed all surgeries. The non-surgery mice from each cage. The 4T1 recordings were one day underwent 15 min of isoflurane anaesthesia only. before inoculation (baseline), at 3 and 24 h, and then In the B16 study mice received a single s/c pre- on days 5, 8, 14, 20 and 22. The B16 mice were filmed at treatment with saline (1 ml/kg), meloxicam (5 mg/kg) baseline, 1 h and on days 5, 9, 13 and 17. or buprenorphine (0.2 mg/kg) just prior to restraint in a 50 ml syringe cartridge modified to expose the tail. Imaging They then received an intravenous (i/v) injection of 100 ml of DPBS containing 5 105 B16-F10-luc-G5 D-Luciferin (PerkinElmer, Beaconsfield, UK) was dis- melanoma cells via the tail. solved in DPBS to 15 mg/ml and frozen at 80C. Mice All mice recovered in a warming cabinet set at 35 were anaesthetised with isoflurane in batches of three 1C for 30 minutes before being returned to the and imaged in an IVIS Spectrum 200 (PerkinElmer, animal holding room. Apart from when mice were Beaconsfield, UK) on a stage maintained at 36C. imaged, all data were collected in the holding room Anaesthesia was provided by face-mask delivery Lofgren et al. 355

of 1.5–2% isoflurane in 0.5 l/min oxygen. Once thawed required Log10 transformation before undergoing to room temperature, 150 mg/kg luciferin was injected ANOVA. Figure 3(a) therefore shows back-trans- s/c. Peak reactivity occurred 12 min later, so after this formed means with 95% confidence intervals open filter scans were taken at 7, 12, 15, 19 and 22 days (95%CI). In the B16 study the raw behaviour data in the 4T1 study. The B16 mice were imaged at 24 h and were acceptable. Living Image software (PerkinElmer, on days 5, 9, 13, 17 and 19. Beaconsfield, UK) quantified tumour burden as the Total Flux (TF; photons/sec/mW/cm2) of biolumines- End-point criteria/terminal assessment cent signals within auto-generated (2% threshold) regions of interest (ROIs) over the mammary or lung Mice were weighed daily and examined according to regions, with additional ROIs over the abdomen to United Kingdom Coordinating Committee for Cancer detect any other metastases. TF on the last day Research (UKCCCR) guidelines.23 The end-point was provided a measure of 4T1 final burden; however, as reached if there was >20% body weight loss alongside signals emerged by day seven it was also possible to poor coat condition and mobility. If these signs were estimate the average growth rate as the change in TF present, after a final IVIS scan mice were to be eutha- from day seven to the final scan on day 22 ((TF on day nased by cervical dislocation without recovery from 22 – TF on day 7)/15). No signals were apparent in the anaesthesia. Any remaining B16 or 4T1 mice were to B16 study until day 17, which was two days before the be euthanased on days 19 or 22 respectively. All mice final scan, so only the final TF values were assessed. underwent a necropsy with ex-vivo imaging of primary The 4T1 ex-vivo primary tumour burden (volume) was tumours, kidney, liver, intestines and lungs. The 4T1 calculated from the caliper measurements (4/3*II*r3). primary tumours were removed and measured with cali- Bivariate correlation analysis (Pearson’s R) was used pers (Mitutoyo, UK Ltd). In the 4T1 study 12 mice had to determine how well the imaging data predicted true to be excluded due to tumour inoculation error, which tumour burden. Apart from the 4T1 growth data resulted in the rapid development of carcinomatosis. (Figure 4(a)) all imaging data, including in the B16 There were no correlations between these occurrences study were Log10 transformed before applying and treatment or surgery. Three B16 mice were also ANOVA. Figures 4(b) and 4(c) therefore show back- excluded, two due to injection error, and one that devel- transformed means þ95%CI. All other results are oped a very large ovarian metastasis. Apart from these mean values 1SEM. The B16 weight data underwent no other mice were euthanased early and the exclusions ANOVA as total change from baseline. The 4T1 body- left sufficient final numbers (shown in Figure 1). weight data were non-homogeneous so percentage weight changes were used. Original data are available Data analysis from an online repository (http://figshare.com). All statistical analyses were performed using the Statistics Package for the Social Sciences (SPSS soft- Results ware version 22.0, SPSS Inc., Chicago, USA). The 4T1 study two cohorts in the 4T1 study had to be analysed separately. Repeated measures ANOVA with probability Body weight: Surgery and multiple doses of bupre- corrected multiple comparisons (Bonferroni) was used. norphine was associated with loss in body In the 4T1 study the between-subjects factors were weight. Six mice were excluded, having lost between ‘drug’, ‘surgery’ and ‘cancer’ status, and ‘time’ was 11 and 24% of baseline weight by day one (>3 times the within-subjects factor. The B16 analysis included the group average). They were all in the cohort 1, one the factors ‘drug’ and ‘time’ only. The baseline eVF from each drug-treated non-surgery cancer group, two thresholds were highly uniform, so to balance the from the saline non-cancer surgery group and two analysis the missing 4T1 values were replaced with the cancer-treated mice that received meloxicam before sur- corresponding group mean. The 4T1 eVF data showed gery. This reduced group sizes to six or seven, which no baseline left or right paw bias so values were aver- was considered acceptable. Over the first three days the aged at each time-point. The 4T1 Hargreaves data single-dose surgery groups lost weight whereas the non- showed a clear paw bias so these data were analysed surgery mice gained, but only 2 versus þ2% (f(1, separately. Automated behaviour analysis software 52) ¼ 9.1, p ¼ 0.004). Neither cancer inoculation nor a (HomeCageScan (HCS): Version 3; Clever Systems single dose of meloxicam or buprenorphine resulted in Inc., USA) was used to obtain the frequency of rearing significant weight change. Mice in the multiple-dose and walking (‘Rear-up’, ‘Come Down’, ‘Remain Rear- cohort lost only negligible weight following surgery. up’ and ‘Walk Left’, ‘Right’ or ‘Slow’) from the video Multiple-dose buprenorphine was associated with material at each time-point. The 4T1 behaviour data 2% greater weight loss over the first three days 356 Laboratory Animals 52(4)

(f(1, 31) ¼ 5.5, p ¼ 0.02). This was more obvious in the only at the 24 h time-point. As the other mice responded non-surgery group, but was not significant. By day six similarly in the days following treatment the responses all mice exceeded baseline weight and showed contin- were pooled within the single- or multiple-dose cancer- ued gains until day 19, with no lasting effects of surgery bearing groups (ignoring surgery) and the two saline, or pre-treatment. Although cancer-bearing mice in each non-cancer control groups. Figures 2(a) (eVF) and 2(b) cohort lost weight as the study ended, this was not sig- (Hargreaves) show the results. The eVF thresholds nificantly more than non-cancer controls. declined more over time in cancer-bearing mice in both the single- and multiple-dose cohorts (‘Time’ (f(5, 49) ¼ 8.4, p < 0.001; f(5, 26) ¼ 21.5, p < 0.001, Nociceptive testing: Multiple doses of buprenorphine respectively), but most obviously spanning days 19–22 significantly increased mechanical thresholds, in mice in the single-dose cohort (Figure 2(a); ‘Time’ whereas cancer growth significantly decreased ‘Cancer’ interaction (f(5, 49) ¼ 3.5, p ¼ 0.008)), indicating them. All groups showed comparable baseline eVF increased nociception as the tumours grew. thresholds. Mice given buprenorphine repeatedly in conjunction with surgery (all of which were cancer inoculated) were the only group that showed significantly Cancer growth reduced the latency to withdraw from increased eVF thresholds (greater force to respond) (f(3, a noxious heat source. Hargreaves latencies were also 28) ¼ 7.5, p ¼ 0.001), that is, decreased nociception, but unaffected by surgery or pre-treatment. Responses from

Figure 2. Nociceptive testing results. (a) Mean (combined paws) electronic von Frey (eVF) thresholds (grams 1SEM) in BALB/c mice in the single- and multiple-dose 4T1 cohorts versus non-cancer controls; showing the significant decline over time in all mice, but more so in cancer groups. (b) Left paw thermal withdrawal thresholds (mean SEM) in the same groups as (a); showing increased thermal sensitivity as tumours grew. (c) eVF threholds (both paws) in the B16 groups where thresholds generally declined during tumour development. (d) Mean thermal (both paws) withdrawal thresholds (SEM) in the B16 groups showing thermal response thresholds were not significantly altered as cancer developed. Significant (Sig.) ‘factors’ from repeated measures ANOVA and p-values are indicated. Lofgren et al. 357 each paw generally occurred sooner over time, but the The lung scanning results reiterated the impact of left paw responded sooner than the right. As a result, surgery, where in both cohorts the surgery groups Figure 2(b) shows data from this paw only with groups showed more extensive lung seeding (single: combined as in Figure 2(a). Mice in the single-dose f(1, 44) ¼ 12.1, p ¼ 0.001; multiple: f(1, 35) ¼ 9, cohort showed a modest latency decrease from baseline p ¼ 0.005, Figure 4(b)). The greatest spread was in the to 24 h (f(1, 53) ¼ 5.5, p ¼ 0.023), indicating decreased groups pre-treated with saline before surgery either as a nociception. After this cancer development was asso- single dose (Sal/S; f(1,16) ¼ 9.7, p < 0.001) or given ciated with a further reduction in response latency repeatedly (Sal/M; f(1,19) ¼ 22.4, p < 0.001). Notably, (‘Time’ ‘Cancer’ interaction; f(6, 318) ¼ 5.6, metastatic signals in the meloxicam or buprenorphine p < 0.001), that is, tumour growth was associated with groups in cohort 1 (Mel/S, Bup/S) were similar to those increased nociception. In the multiple-dose cohort both in the non-surgery groups (p ¼ 0.258, p ¼ 0.23, respect- the left and right paws were sensitised by 24 h (right: ively), and in cohort 2 the surgery/non-surgery groups f(1, 31) ¼ 7.3, p ¼ 0.001; left: f(1.31) ¼ 18.2, p ¼ 0.0001), given buprenorphine repeatedly were almost identical and both paws then responded sooner as tumours grew (Bup/M; p ¼ 0.83). The ex-vivo measurements showed (right: f(6,186) ¼ 2.1, p ¼ 0.049; left: f(6, 186) ¼ 5.3, mice in cohort 1 with the largest tumours had more p < 0.0001), but as the significance values indicated, metastases (Pearson’s r ¼ 0.34, p ¼ 0.024) and more the left paw became the most sensitised. rapid tumour growth over days 7–22 (R2 ¼ 0.4, p < 0.001). Behaviour: While surgery and analgesic treatments did not significantly alter behaviour, spontaneous B16 study active behaviours decreased as tumours grew. Rearing and walking were unaffected by drug Body weight: Mice treated with buprenorphine lost administration or surgery at any point during either slightly more weight than those receiving saline or the single- and multiple-dose 4T1 studies. As a result, meloxicam. The three test groups had similar initial these behaviours were combined to create a summary weights (22.8–23.6 g) and there were no significant variable ‘Behave’ which was used to assess any later losses on day one. By day three mice receiving bupre- effects of cancer development in each cohort of norphine lost more weight than those given saline or mice.22,24 Figure 3(a) shows the activity changes in meloxicam (f(1,27) ¼ 3.8, p ¼ 0.036), but an average of the control groups (saline, non-cancer) versus the only 1 g more. After this the saline and meloxicam cancer-bearing mice in the single- and multiple-dose groups maintained weight and the buprenorphine cohorts. At 3 h all mice were more active than at base- group gained, but only slightly more (1 g). line (f(1, 61) ¼ 15.8, p ¼ 0.001; f(1, 34) ¼ 9.1, p ¼ 0.005, Nevertheless, there was a significant ‘Time’ effect and cohorts 1 and 2, respectively). This dissipated over 24 h a ‘Time’ ‘Pre-treatment’ interaction (f(17, 408) ¼ 5.8, and activity was relatively normal until between days 20 p < 0.001; f(17, 408) ¼ 2.7, p < 0.001, respectively). and 22, where compared with non-cancer controls, There were no significant weight changes as the study tumour-bearing mice in cohort 1 showed a greater ended. activity decline (‘Time’ ‘Cancer’ interaction; f(1, 61) ¼ 7.0, p ¼ 0.011). However, this was not appar- Nociceptive testing: Mechanical thresholds, but not ent in the equivalent cancer-bearing mice in cohort 2. latency to withdraw from thermal stimulus, significantly decreased as cancer developed. Baseline eVF Tumour growth as indicated by IVIS signal intensity: thresholds were similar across the three groups and both Surgery was a more significant tumour growth mod- paws responded similarly (2.7 0.6 g; Figure 2(c)). Pre- ulator than analgesia. Signal intensity increased more treatment had no effect on post-inoculation thresholds rapidly in mice in cohort 1 that underwent surgery which continued to decline as cancer developed (‘Time’ compared with non-surgery controls (20.8 159 significant; f(5, 120) ¼ 30, p < 0.0001). Although there vs. 162 24 109 photons/s/day; f(1, 38) ¼ 24.8, was a partial stabilisation between days five and nine, p < 0.0001, Figure 4(a)), and they also showed stronger thresholds were lower than at baseline at every subse- signals on the last day (f(1, 38) ¼ 25.8, p < 0.001). In quent time-point (p 0.001). The baseline B16 cohort 2 the surgery/non-surgery growth difference Hargreaves latencies were also similar initially, and was somewhat less, but still significantly greater in the although they also declined from baseline to 1 h (from mice that underwent surgery (27.59 33 vs. 123 3.9 0.7 to 3.5 1.1 s; Figure 2(d)), not significantly 26 109 photons/s/day; f(1, 31) ¼ 5.1, p ¼ 0.03). The (p ¼ 0.06). ex-vivo caliper measurements indicated larger volume tumours in mice that underwent surgery (554 50 vs. Behaviour: Buprenorphine significantly increased 420 40mm3; all cancer-bearing groups combined). walking behaviour. Figures 3(b) and 3(c) illustrate 358 Laboratory Animals 52(4)

Figure 3. Behaviour results. (a) The geometric mean frequency (1SEM) of the summary measure ‘Behave’ (þ95% Confidence Intervals (CI)) in mice in the 4T1 single- or multiple-dose cohorts versus non-cancer single-dose controls, illustrating the post-procedural activity enhancement but relatively normal behaviour, even when cancer was well developed. The mean frequency (SEM) of walking (b) or rearing (c) in the B16 groups; illustrating that at 1 h mice walked more following s/c saline (1 ml/kg) or meloxicam (10 mg/kg), but especially more after buprenorphine (0.2 mg/kg) compared with baseline (**: p < 0.001(b)). All mice also reared more at 1 h (**: p < 0.001(c)) except those that received buprenorphine. The effects of buprenorphine and those in the meloxicam and saline groups subsided by five days and behaviour was subsequently normal. rearing and walking frequency separately because they until day 17, but after this development occurred were affected by buprenorphine differently. Overall, rapidly. There was no difference in final day signal both were significantly increased at 1 h following intensity between mice that had received saline or tumour inoculation (rearing: f(1, 26) ¼ 151, p < 0.001; buprenorphine, but as Figure 4(c) shows, signal inten- walking: f(1, 26) ¼ 58, p < 0.001); however, relative to sity was lower in mice that received meloxicam the behaviour of mice in the meloxicam or saline (p ¼ 0.014, p ¼ 0.01; meloxicam vs. saline or buprenor- groups, at the 1 h time-point pre-treatment with phine, respectively). Neither drug affected abdominal buprenorphine was associated with increased walking metastases. (Figure 3(b); f(2, 26) ¼ 24.6, p < 0.001) and less rearing (Figure 3(c); f(2, 26) ¼ 13.5, p < 0.001). Both rearing Discussion and walking became normal by day five and remained as such until the study ended. Out of concern that analgesics can alter tumour development, animal cancer studies often withhold them. Tumour growth, as indicated by IVIS signal intensity: However, this ignores the possibility that cancer-related Meloxicam, but not buprenorphine, reduced lung pain or distress could cause unnecessary scientific vari- metastases. There was no significant change in TF ation. Two studies were undertaken to assess whether Lofgren et al. 359

Figure 4. Imaging results. (a) Mean rate of development of signals from 4T1 primary tumours over days 7–22 (TF 109 photons/s/cm2/day SEM) in mice that underwent surgery or not, illustrating the significant greater surgery-related tumour growth rate in both 4T1 cohorts (**: p < 0.001: *: p < 0.05). (b) Geometric mean TF (photons/s/cm295% CI) of ex- vivo metastatic lung signals in mice in the single (S) or multiple (M) dose 4T1 cohorts pre-treated with saline (Sal; 1 ml/kg), meloxicam (Mel; 10 mg/kg) or buprenorphine (Bup; 0.2 mg/kg) before undergoing surgery or not. In the multiple- and single-dose saline groups (Sal/S or Sal/M) surgery significantly increased lung metastases (**: p < 0.001), but buprenorphine given singly and especially repeatedly (Bup/S, Bup/M) protected against greater tumour spread (buprenorphine- treated surgery vs. non-surgery groups, p ¼ NS). (c) The geometric mean TF (photons/s/cm295% CI) on the last day in the B16 study, illustrating meloxicam pre-treatment significantly reduced lung seeding compared with saline or buprenorphine (*: p < 0.05). buprenorphine or meloxicam altered tumour growth in would show behaviour changes and nociceptive sensi- BALB/c mice developing 4T1 mammary carcinoma, tivity changes suggesting underlying pain, but that and in C57BL/6NCrl mice inoculated with B16 melan- these effects would be minimised in the groups pre-trea- oma. Both studies used luciferase-expressing cells lines ted with meloxicam or buprenorphine. Neither the B16 so tumour growth and spread could be determined or 4T1 inoculation procedures appeared to cause sig- using bioluminescent imaging. The 4T1 study included nificant pain, and there was also little evidence of groups undergoing tumour inoculation in conjunction pain as each type of cancer developed. It was predicted with surgery. A secondary aim was to assess any pain that the B16 inoculation and 4T1 percutaneous inocu- the inoculation procedures might cause, and whether lation procedure would be relatively harmless, but pain occurred as tumours grew. This was assessed the relatively benign effect of undertaking 4T1 inocula- using body weight and behaviour changes, and by tion paired with surgery was unexpected. The main recording parallel signs of nociceptive responding via findings were that surgery significantly increased 4T1 thermal and mechanical nociceptive threshold testing. tumour growth and metastatic spread, but provision It was hypothesised that mice undergoing surgery of pre- and post-operative buprenorphine neutralised 360 Laboratory Animals 52(4) the impact of surgery on the tumour model. Providing The only obvious behavioural change in either study peri-procedural analgesia was therefore shown to be was in the buprenorphine-treated B16 mice, which beneficial, by more closely approximating tumour showed a substantial initial increase in walking and growth to the spontaneous development experienced lack of rearing. These were most probably non-specific by human patients that this research is designed to effects as previously seen in buprenorphine-treated mice model. following either vasectomy or laparotomy.24,29 In gen- The BALB/c mice in the 4T1 study were predicted to eral, the activity increases following B16 inoculation show an impact of surgery comparable with that in may have reflected the normal tendency of mice to another of our recent investigations in which BALB/c explore a novel environment, in this case the filming mice underwent laparotomy but showed substantially cage. Although hyper-locomotion can be caused by reduced behavioural activity and significant post- chronic stress,30,31 it is again doubtful this could have surgical weight losses.22 However, on this occasion been the case following the short period of restraint they generally became more active following either needed for B16 inoculation. anaesthesia or surgery and maintained body weight. Nociceptive responding continued to increase as Albeit the previous BALB/c mice were male, the litera- 4T1 tumours grew (Figure 2(a) and 2(b)), and in the ture on sex-related pain sensitivity is highly non- B16 study there was a less obvious but still significant consensual. The most recent knowledge also suggests reduction in mechanical thresholds as the study ended females should be more, rather than less, prone to (Figure 2(c)). The nociceptive threshold was reduced, pain,25 so although it cannot be discounted, sex was indicating that the mice experienced greater pain. thought unlikely to account for such opposing post- However, by the time these changes were most obvi- surgical outcomes. More relevantly perhaps, the aim ous, the mice in both studies had been exposed to of the previous study was to validate use of an agent numerous different potential stressors. Apart from that was supposed to enable imaging of inflammation. being isolated from cage-mates during nociceptive test- The laparotomy procedure was therefore modified such ing and filming, they underwent repetitive anaesthesia that after the agent was injected it included 60 s of vis- for imaging, daily weighing and a health inspection, ceral abrasion. These additional manipulations may and in the 4T1 study also restraint for tumour palpa- have previously been aggravating to normal behaviour, tion. It is possible that the combined impact of these and it was possibly also relevant that the two studies experiences was sufficient to cause some degree of had different surgeons. Whereas the previous study hyperalgesia. In a previous study, mice with bladder used mattress sutures, in this study a continuous sub- cancer20 demonstrated evidence of pain by showing an cuticular suture was used to close the skin. This may enhanced conditioned place preference for morphine have been a refinement that helped to limit alterations as their tumours grew. Here, the B16 mice behaved in behaviour, but it is impossible to verify whether these relatively normally; their mechanical response thresh- protocol variations contributed to the lesser impact of olds showed only a modest decrease as cancer devel- surgery found here. An obvious limitation of this study oped, and there were no significant thermal response is that we did not have the opportunity to replicate the alterations. As the 4T1 study ended, increased noci- study to determine if the pain responses measured were ceptive sensitivity was accompanied by a minor reduc- reproducible. tion in behavioural activity confined to mice in cohort The other noticeable effect of surgery was that 1 and not those bearing the same cancer in cohort 2 both thermal and mechanical sensitivity had increased (Figure 3(a)). at 24 h. However, this change was also apparent in Imaging was used to determine any drug-related 4T1 non-surgery controls, indicating pain alone tumour growth changes. Although meloxicam reduced may not have been the source of this sensitivity. B16 lung seeding as compared with saline or buprenor- Alterations as a result of drug treatment were phine, buprenorphine had no effect on this model of evidenced by the increase in 4T1 mechanical thresholds metastatic growth (Figure 4(c)), and neither analgesic at 24 h following multiple-dose buprenorphine; how- had any detrimental impact on 4T1 tumour growth ever, this was only seen following surgery and not, as consistency or metastases. However, surgery caused would have been predicted, in the equivalent non-sur- 4T1 tumours to develop more rapidly and spread gery group. Finally, stress causes analgesia; but it is more (Figure 4(a) and 4(b)). Such changes have previ- severe or prolonged the opposite can occur, that is, ously been attributed to removal of anti-angiogenesis hyperalgesia.26–28 However, while the stress of the sur- control, growth factor release, or metabolic, neuroen- gery may have been sufficient to cause hyperalgesia, the docrine and immunological suppression.6,11,13 Several relatively minor procedure used in the B16 investigation previous studies have also shown stress due to anaes- was unlikely to be sufficient to induce this level of thesia, pain or hypothermia12,32–34 can promote many stress. types of cancer.10–16 Although it is unknown which Lofgren et al. 361 aspect contributed most to the present 4T1 acceler- Centre, Newcastle University, Framlington Place, Newcastle ation, because it was partially neutralised by buprenor- upon Tyne, NE2 4HH, UK. phine, stress again seems the most likely candidate. While surgery alone led to an increase in lung metas- Declaration of Conflicting Interests tases (Figure 4(b)), this was prevented if mice had repeated peri-operative doses of buprenorphine at a The author(s) declared no potential conflicts of interest with dose commonly used for pain prevention.35 Previous respect to the research, authorship, and/or publication of this article. studies have found analgesics can reduce surgery-associated increases in tumour recurrence,12,13 and there are also examples where buprenorphine had either no Funding 8,9 impact or supported normal tumour growth. The author(s) disclosed receipt of the following financial sup- Perhaps due to these aspects and its post-surgical port for the research, authorship, and/or publication of this 9 stress-preventative properties, giving buprenorphine article: The American College of Laboratory Animal was beneficial to model consistency. Although this Medicine (ACLAM Foundation) and the UK National obviously needs confirmation with other cancer Centre for the 3Rs (NC3Rs). models, it suggests that in some cases, especially in cancer studies requiring surgery, that mice might benefit from receiving some form of pain or stress preven- References tion. Therefore, future research goals include similar 1. Workman P, Aboagye EO, Balkwill F, et al. Guidelines evaluations as performed in these studies in additional for the welfare and use of animals in cancer research. Br J orthotopic tumour models requiring surgery. Cancer 2010; 102: 1555–1577. Furthermore, a meta-analysis of the relative success 2. Loi M, Di Paolo D, Becherini P, et al. The use of the orthotopic model to validate antivascular therapies for of anti-neoplastic therapies developed in mouse cancer. Int J Dev Biol 2011; 55: 547–555. tumour models requiring and not requiring surgery, 3. Carbone L and Austin J. Pain and laboratory animals: as well as in studies providing or not providing peri- Publication practices for better data reproducibility and operative analgesia, would allow further exploration of better animal welfare. PLoS ONE 2016; 11: e0155001. the translation of the results of murine orthotopic 4. Richardson CA and Flecknell PA. Anaesthesia and post- tumour models to human health. operative analgesia following experimental surgery in Mammary tumours most commonly occur in laboratory rodents: Are we making progress? Altern females and therefore we chose to assess the 4T1 Lab Anim 2005; 33: 119–127. tumours in female mice. However, it might be asked 5. Connolly EM, Harmey JH, O’Grady T, et al. Cyclo- why the B16 mice were also female. Although this oxygenase inhibition reduces tumour growth and metas- has been dealt with before,20 it was to answer the tasis in an orthotopic model of breast cancer. Br J Cancer need for studies that do not only consider males. A 2002; 87: 231–237. minor point to mention was the good agreement 6. Roche-Nagle G, Connolly EM, Eng M, et al. Antimetastatic activity of a cyclooxygenase-2 inhibitor. between the caliper measurements and the imaging Br J Cancer 2004; 91: 359–365. data. Although manual tumour burden estimates are 7. Leahy KM, Ornberg RL, Wang Y, et al. Cyclooxygenase- generally inaccurate, in this case they partially pre- 2 inhibition by celecoxib reduces proliferation and dicted eventual 4T1 tumour spread. induces apoptosis in angiogenic endothelial cells in vivo. These studies document minimal impacts of anal- Cancer Res 2002; 62: 625–631. gesia on tumour growth in at least one cancer model. 8. Martucci C, Panerai AE and Sacerdote P. Chronic fen- Minimising surgical stress via any means possible seems tanyl or buprenorphine infusion in the mouse: Similar a logical refinement that could remove some current analgesic profile but different effects on immune barriers to precautionary analgesic use in animal responses. Pain 2004; 110: 385–392. cancer studies. While possibly less critical in basic sci- 9. Franchi S, Panerai AE and Sacerdote P. Buprenorphine ence research, pre-clinical studies intended to directly ameliorates the effect of surgery on hypothalamus-pitui- model benefits for human patients may arguably be tary-adrenal axis, natural killer cell activity and meta- best served by treating animals similarly to human static colonization in rats in comparison with morphine or fentanyl treatment. Brain Behav Immun 2007; 21: cancer patients, where analgesic use is normal and usu- 767–774. ally beneficial. 10. Beilin B, Shavit Y, Trabekin E, et al. The effects of postoperative pain management on immune response to sur- Acknowledgements gery. Anesth Analg 2003; 97: 822–827. Thanks are due to Liz Matheson for cell culturing and to 11. Gottschalk A, Sharma S, Ford J, et al. Review article: Charles River UK for donating the mice. Work was carried The role of the perioperative period in recurrence after out at Institute of Neuroscience, Comparative Biology cancer surgery. Anesth Analg 2010; 110: 1636–1643. 362 Laboratory Animals 52(4)

12. Snyder GL and Greenberg S. Effect of anaesthetic tech- buprenorphine treatment on faecal corticosterone con- nique and other perioperative factors on cancer recur- centrations and behaviour assessed by manual and auto- rence. Br J Anaesth 2010; 105: 106–115. mated analysis methods in C57 and C3H mice. PLoS 13. Benish M and Ben-Eliyahu S. Surgery as a double-edged ONE 2013; 8: e75948. sword: A clinically feasible approach to overcome the 25. Sorge RE, Mapplebeck JC, Rosen S, et al. Different metastasis-promoting effects of surgery by blunting immune cells mediate mechanical pain hypersensitivity stress and prostaglandin responses. Cancers (Basel) in male and female mice. Nat Neurosci 2015; 18: 2010; 2: 1929–1951. 1081–1083. 14. Lee JW, Shahzad MM, Lin YG, et al. Surgical stress 26. Gameiro GH, Andrade AdS, de Castro M, et al. The promotes tumor growth in ovarian carcinoma. Clin effects of restraint stress on nociceptive responses induced Cancer Res 2009; 15: 2695–2702. by formalin injected in rat’s TMJ. Pharmacol Biochem 15. Page GG, Blakely WP and Ben-Eliyahu S. Evidence that Behav 2005; 82: 338–344. postoperative pain is a mediator of the tumor-promoting 27. Gameiro GH, Gameiro PH, Andrade AdS, et al. effects of surgery in rats. Pain 2001; 90: 191–199. Nociception- and anxiety-like behavior in rats submitted 16. Hasegawa H and Saiki I. Psychosocial stress augments to different periods of restraint stress. Physiol Behav tumor development through beta-adrenergic activation 2006; 87: 643–649. in mice. Jap J Cancer Res 2002; 93: 729–735. 28. Rivat C, Laboureyras E, Laulin J-P, et al. Non-nocicep- 17. Page GG and Ben-Eliyahu S. Indomethacin attenuates tive environmental stress induces hyperalgesia, not anal- the immunosuppressive and tumor-promoting effects of gesia, in pain and opioid-experienced rats. surgery. J Pain 2002; 3: 301–308. Neuropsychopharmacology 2007; 32: 2217–2228. 18. Flecknell P. Analgesia and post-operative care. In: 29. Bourque SL, Adams MA, Nakatsu K, et al. Comparison Laboratory Animal Anaesthesia (Fourth Edition). of buprenorphine and meloxicam for postsurgical anal- Boston: Academic Press, 2016, pp.141-192. gesia in rats: Effects on body weight, locomotor activity, 19. Rashid OM, Nagahashi M, Ramachandran S, et al. An and hemodynamic parameters. J Am Assoc Lab Anim Sci improved syngeneic orthotopic murine model of human 2010; 49: 617–622. breast cancer progression. Breast Cancer Res Treat 2014; 30. Strekalova T, Spanagel R, Dolgov O, et al. Stress- 147: 501–512. induced hyperlocomotion as a confounding factor in anx- 20. Roughan JV, Coulter CA, Flecknell PA, et al. The con- iety and depression models in mice. Behav Pharmacol ditioned place preference test for assessing welfare conse- 2005; 16: 171–180. quences and potential refinements in a mouse bladder 31. Zimprich A, Garrett L, Deussing JM, et al. A robust and cancer model. PLoS ONE 2014; 9: e103362. reliable non-invasive test for stress responsivity in mice. 21. Kim J-B, Urban K, Cochran E, et al. Non-invasive detec- Front Behav Neurosci 2014; 8: 125. tion of a small number of bioluminescent cancer cells 32. Arranz A, Venihaki M, Mol B, et al. The impact of stress in vivo. PLoS ONE 2010; 5: e9364. on tumor growth: peripheral CRF mediates tumor-pro- 22. Roughan JV, Bertrand HG and Isles HM. Meloxicam moting effects of stress. Mol Cancer 2010; 9: 261. prevents COX-2-mediated post-surgical inflammation 33. Sloan EK, Priceman SJ, Cox BF, et al. The sympathetic but not pain following laparotomy in mice. Eur J Pain nervous system induces a metastatic switch in primary 2015; 23: 712. breast cancer. Cancer Res 2010; 70: 7042–7052. 23. UKCCCR. United Kingdom Co-ordinating Committee 34. Androulidaki A, Dermitzaki E, Venihaki M, et al. on Cancer Research (UKCCCR) Guidelines for the Corticotropin releasing factor promotes breast cancer Welfare of Animals in Experimental Neoplasia (Second cell motility and invasiveness. Mol Cancer 2009; 8: 30. Edition). Br J Cancer 1998; 77: 1–10. 35. Guarnieri M, Brayton C, DeTolla L, et al. Safety and 24. Wright-Williams S, Flecknell PA and Roughan JV. efficacy of buprenorphine for analgesia in laboratory Comparative effects of vasectomy surgery and mice and rats. Lab Anim (NY) 2012; 41: 337–343.

Re´sume´ Les mode`les orthotopiques murins de cancer exigent souvent une chirurgie, laquelle peut potentiellement causer douleur et de´tresse, mais les analge´siques sont souvent exclus car ils peuvent alte´rer le de´veloppement des tumeurs. Deux cancers orthotopiques implante´s ont e´te´ e´tudie´s chez des souris preálablement traiteés par me´loxicam (10 mg/kg), bupreńorphine (0.2 mg/kg) ou une solution saline (1 ml/kg). Les tumeurs ont e´te´ imageés et le bien-eˆtre social a e´te´ e´value´ en mesurant le poids du corps, le comportement et les re´ponses nociceptives. Dans l’e´tude 1, les souris BALB/c ont e´te´ inoculeés avec un carcinome mammaire 4T1 ou une solution saline pendant la chirurgie ou l’anesthe´sie. Le pre´traitement avec une dose unique de bupreńorphine s’e´tant re´ve´le´ beńe´fique pour la constance de la croissance du cancer, une deuxie`me cohorte de souris a reçuen plus une solution saline ou de la bupreńorphine a` 12 et 24 heures. L’ope´ration a entraıˆne´ une augmentation de la croissance des tumeurs mammaires et des me´tastases pulmonaires. Ces effets Lofgren et al. 363 inde´sirables ont e´te´ atteńue´s par la bupreńorphine utiliseé en pre´traitement, surtout lorsqu’elle e´tait admin- istreé de façon re´pe´teé. Les souris porteuses de tumeurs mammaires sont devenues moins actives et les seuils nociceptifs ont diminue´ au fil du temps, indiquant la pre´sence d’un certain malaise au fur et a` mesure que les tumeurs augmentaient. Dans l’e´tude 2, des souris C57BL/6 ont e´te´ inoculeés par un me´lanome B16. Ce mode`le non-chirurgical a e´te´ utilise´ pour de´terminer si l’ibuprofe`ne ou la bupreńorphine affectaient l’ensemencement des poumons par le cancer. Bien que le me´loxicam ait re´duit l’ensemencement des poumons par le me´lanome B16, la bupreńorphine n’a pas eu cet effet. Les seuils mećaniques ont diminue´ au fur et a` mesure que le cancer s’est de´veloppe´ chez les souris porteuses du me´lanome, mais l’ampleur de cette diminution s’est ave´reé insuffisante pour conclure a` l’existence de preóccupations en matie`re de bien-eˆtre. Cette e´tude souligne la valeur scientifique de l’utilisation de mode`les non-chirurgicaux, dans la mesure du possible. Lorsque la chirurgie doit eˆtre effectueé au moment de l’inoculation de la tumeur, ses effets dev- raient eˆtre controˆle´s par des analge´siques afin d’accroıˆtre la valeur et peut-eˆtre la translation de la recherche.

Abstract Orthotopische Maus-Krebs-Modelle bedu¨rfen oft chirurgischer Eingriffe und verursachen potenziell Schmerz oder Leid. Analgetika werden jedoch haüfig nicht verabreicht, da sie u. U. die Tumorentwicklung vera¨ndern ko¨nnen. Zwei orthotopisch implantierte Karzinome wurden bei Maüsen untersucht, die zuvor mit Meloxicam (10 mg/kg), Buprenorphin (0.2 mg/kg) oder Salzlo¨sung (1 ml/kg) behandelt worden waren. Tumore wurden bildlich dargestellt und das Wohlbefinden wurde anhand von Ko¨rpergewicht, Verhalten und nozizeptiven Reaktionen bewertet. In Studie 1 wurden BALB/c Maüse wa¨hrend der Operation oder Ana¨sthesie mit 4T1 Mammakarzinom oder Salzlo¨sung inokuliert. Da die Vorbehandlung mit einer Einzeldosis Buprenorphin einem gleichbleibenden Krebswachstum fo¨rderlich schien, erhielt eine zweite Maüsekohorte zusa¨tzlich Salzlo¨sung oder Buprenorphin nach 12 und 24 Stunden. Die Operation resultierte in erho¨htem Mammatumorwachstum und Lungenmetastasen. Diese unerwu¨nschten Folgen wurden durch Vorbehandlung mit Buprenorphin verringert, insbesondere bei wiederholter Verabreichung. Mammatumor tragende Maüse wurden inaktiver und nozizeptive Schwellen sanken mit der Zeit als Anzeichen fu¨r gewisses Unbehagen angesichts zunehmenden Tumorwachstums. In Studie 2 erhielten C57BL/6-Maüse B16-Melanom. Dieses nicht-chirurgische Modell wurde verwendet, um zu ermitteln, ob Meloxicam oder Buprenorphin die Krebsbesiedlung der Lunge beeinflussten. Meloxicam reduzierte die B16-Lungenbesiedlung, Buprenorphin jedoch nicht. Mechanische Schwellen sanken mit zunehmender Karzinomentwicklung bei Melanom tragen- den Maüsen, jedoch in einem Ausmaß, das unzureichend war, um schlussfolgern zu ko¨nnen, dass signifikante Beeintra¨chtigungen des Wohlbefindens zu befu¨rchten sind. Diese Studie unterstreicht den wissenschaftlichen Wert des Einsatzes nicht-chirurgischer Modelle, soweit mo¨glich. Wenn zur Tumorinokulation eine Operation erfolgen muss, sollten die Auswirkungen mit entsprechenden Analgetika kontrolliert werden, um den Wert und mo¨glicherweise die U¨berleitung der Forschungsergebnisse zu optimieren.

Resumen Los modelos murinos con cańcer ortoto´pico a menudo requieren cirugıá, lo que suele provocar dolor o malestar. No obstante, los analge´sicos a menudo son negados ya que pueden alternar el desarrollo del tumor. Se investigaron dos cańceres implantados ortoto´picamente en ratones que habıán recibido meloxicam (10 mg/kg), buprenorfirna (0.2 mg/kg) o salina (1 ml/kg). Los tumores fueron escaneados y se analizo´ el bienestar utilizando el peso corporal, el comportamiento y las respuestas nociceptivas. En el estudio 1, los ratones BAB/c fueron incoculados con carcinoma de mamas 4T1 o salina durante cirugıá o anestesia. Ya que un tratamiento previo con una uńica dosis de buprenorfina parecio´ ser beneficioso para la consistencia de crecimiento del cańcer, un segundo cohorte de ratones tambień recibio´ salina o buprenorfina a las 12 y 24 horas. La cirugıá provoco´ un crecimiento del tumor de mama y una meta´stasis pulmonar. Estos efectos no deseados fueron aliviados con un tratamiento previo con buprenorfina, especialmente al administrarse repe- tidamente. Los ratones con tumor de mama fueron menos activos y los lı´mites nociceptivos decrecieron con el tiempo, lo cual indicaba un malestar a medida que los tumores crecıán. En el estudio 2, los ratones C57BL/ 6 recibieron melanoma B16. Este modelo no quiru´rgico fue utilizado para determinar si el meloxicam o la buprenorfina afectaban la germinacioń del cańcer en los pulmones. Mientras que el meloxicam redujo la 364 Laboratory Animals 52(4) germinacioń pulmonar B16, la buprenorfina no lo hizo. Los lı´mites mecańicos decrecieron a medida que el cańcer se desarrollaba en los ratones con melanomas, pero la magnitud de esto fue insuficiente para concluir que habıá una preocupacioń significativa por el bienestar de los animales. Este estudio destaca el valor cientı´fico que tiene utilizar modelos no quiru´rgicos, siempre que sea posible. Cuando se debe realizar inter- venciones quiru´rgicas en el momento de la inoculacioń del tumor, los efectos deberıán controlarse con analge´sicos adecuados para aumentar el valor y la posible implementacioń del estudio. laboratory an imals Original Article limited Laboratory Animals 2018, Vol. 52(4) 365–372 ! The Author(s) 2017 Direct comparison of vasectomized Reprints and permissions: sagepub.co.uk/ males and genetically sterile journalsPermissions.nav DOI: 10.1177/0023677217748282 Gapdhs knockout males for the journals.sagepub.com/home/lan induction of pseudopregnancy in mice

Wiebke Garrels1, Dirk Wedekind1, Isabell Wittur1, Ulrike Freischmidt1, Dirk Korthaus2, Thomas Ru¨licke2 and Martina Dorsch1

Abstract The laboratory mouse is the most used animal model in biomedical research. Several artificial reproductive techniques, such as revitalization of cryopreserved strains, rederivation after hygienic contaminations and the production of transgenic mouse models, require the transfer of preimplantation embryos to surrogate mothers. Pseudopregnancy is essential in recipient females and is induced by mating with sterile males. Commonly, surgically vasectomized males are used for this purpose. As an alternative, genetically modified mouse strains have been identified, in which homozygous infertile males are sexually active. Here, we investigated the suitability of genetically infertile Gapdhstm1Dao males under routine laboratory conditions with respect to plug rates, pregnancy rates and frequency of born offspring after embryo transfer. Our results showed no significant differences for these aspects between Gapdhstm1Dao and vasectomized CD2F1 males. In addition, we evaluated the efforts to obtain a defined number of sterile males either by breeding of sterile mutants or surgical vasectomy, and addressed the impact of both options on animal welfare. In conclusion, infertile males of the Gapdhstm1Dao line are a reliable alternative to vasectomized males for the induction of pseudopregnancy, and can contribute to the refinement of the procedure by avoiding surgical interventions.

Keywords mice, vasectomy, pseudopregnancy, embryo transfer

Date received: 30 June 2017; accepted: 22 November 2017

Introduction pseudopregnancy needs an ejaculation and cannot be Embryo transfer is an essential method for reproduct- achieved by male intromission or mechanical stimula- ive technologies in mice, i.e. for generation of genetic- tion of the cervix alone.3 ally modified strains, for saving of lines with problems Recipient females for embryo transfer have to be in fertility or raising of litters, for in-vitro fertilization, mated with infertile males to induce uterine differenti- for rederivation of hygienically contaminated strains or ation resulting in the receptive state for the implant- for revitalization of cryopreserved embryos. ation of the transferred embryos. Commonly, infertile Availability of pseudopregnant surrogate mothers is males for the induction of pseudopregnancy are an essential requirement for the performance of a successful embryo transfer. In mice, the establishment of a 1Institute for Laboratory Animal Science, Hannover Medical pregnancy requires a vaginal-cervical stimulation. School, Germany During copulation the male produces a vaginal plug 2Institute of Laboratory Animal Science, University of Veterinary which is formed by secretes of male accessory glands. Medicine Vienna, Austria This copulatory plug not only seals the vagina but also 1,2 Corresponding author: induces permanent pressure to the cervix. The vagi- M Dorsch, Institute for Laboratory Animal Science, Hannover nal plug indicates that intromission and ejaculation on Medical School, Carl-Neubergstr. 1, 30625 Hannover, Germany. the male has occurred. In mice, the induction of Email: [email protected] 366 Laboratory Animals 52(4) produced by vasectomy under general anaesthesia. Whenever possible, mice were kept in sibling groups. The severity of the intervention is classified as a ‘mod- The microbiological status was examined as recom- erate’ procedure. After surgery, sterility has to be con- mended by FELASA and absence of listed microorgan- firmed by test mating, which can be started after a isms,7 except Pasteurella pneumotropica, Helicobacter recovery period of at least three weeks to ensure that sp. and norovirus, was confirmed. no stored sperm cells were left in the ductus deferens. The experiments were in accordance with the Alternatively, genetically modified strains in which German Animal Welfare Legislation and approved by the genetic modification leads to male infertility in the local Institutional Animal Care and Research the homozygous carriers have been reported for this Advisory Committee and permitted by the Lower purpose in recent years which could be useful for the Saxony State Office for Consumer Protection and induction of pseudopregnancy.4–6 For example, the Food Safety (reference number 42500/1H and 13A404). suitability of Prm1-EGFP overexpressing males was Mice of the Gapdhstm1Dao line, kindly provided by Dr tested and discussed for the production of pseudopreg- D O’Brein, University of North Carolina, USA, were nant surrogate mothers.6 To the best of the authors’ on a mixed 129S6;B6 background. After backcrossing knowledge, however, our study is the first that covers to C57BL/6N the mating habits of males deteriorated not only the induction of pseudopregnancy, but also considerably. To achieve better reproductive perform- several other important aspects of the breeding and ance the mutants were backcrossed for four generations use of sterile mutant males. to Crl:CD1 (ICR) outbred mice. Animals of the N4 In the presented study, we assessed the suitability of backcross generation were intercrossed to produce infertile Gapdhstm1Dao knockout males for large-scale homozygous females which have been used henceforth production of pseudopregnant surrogates under routine to maintain the colony by mating with heterozygous laboratory conditions.4 In Gapdhstm1Dao mice the glycer- males. Apart from the infertility of homozygous aldehyde-3-phosphate dehydrogenase spermatogenic Gapdhstm1Dao knockout males, no further pathological gene (Gapdhs) was deleted by gene targeting via hom- findings have been described. The knockout males pro- ologous recombination in TC-1 embryonic stem cells. duce normal sperm count, behave like wildtype males The mating behaviour of homozygous Gapdhs knockout when mated and produce a prominent vaginal plug. males is comparable to wildtype males including the pro- Spermatozoa from Gapdhs-/- males show significantly duction of a prominent vaginal plug. Due to low ATP decreased motility, probably due to very low ATP levels, sperm from Gapdhs-/- males have a significant levels, resulting in complete infertility.4 In contrast, decreased motility which results in complete infertility. Gapdhs-/- females as well as heterozygous Gapdhsþ/- We compared plug rates, pregnancy rates after embryo males and females do not differ from wildtype mice in transfer, and the portion of transferred embryos that their reproductive success. DNA for genotyping of developed to term (birth rate) after mating of mutant offspring was isolated from ear punches taken B6CBAF1 hybrid females with either surgically vasecto- for marking the animals. Details for genotyping can be mized CD2F1 hybrid males, or infertile Gapdhs mutants found under: https://www.mmrrc.org/catalog/sds.php? with CD1 outbred background. In addition, we calcu- mmrrc_id¼36555. The observed inheritance pattern of lated/evaluated the required efforts to obtain a defined the mutation was according to Mendel’s laws. number of sterile males, i.e. the breeding effort, cage CD2F1 males were generated by mating BALB/ space, effort for genotyping and surgery. Moreover, we cJHanZtm (C) females and DBA/2JHanZtm (D2) also addressed animal welfare aspects of both males. F1-males were vasectomized at an age of 6 weeks. approaches with respect to the 3R guidelines. B6CBAF1/Rj females were purchased from Janvier (www.janvier-breedingcenter.com) at an age of four weeks. They were used as embryo transfer recipients Material and methods at an age of 8–10 weeks. Animals and husbandry Anaesthesia and analgesia for surgery All animals were kept in an individually ventilated caging system (IVC, BioZone, Chester, UK) in cages with a Anaesthesia was applied by intraperitoneal (i.p.) injec- ground area of 440 cm2 under controlled standardized tion of a mixture of 80 mg/kg ketamine 10% (KetanestÕ, conditions: temperature 21 2C, relative humidity Bayer Leverkusen, Germany) 2 mg/kg xylazine 2% 50 5% and artificial light (14 h light, 10 h dark, light (RompunÕ, Bayer, Leverkusen, Germany), and 0.7 mg/ on at 06:00 a.m.). Sterilized commercial softwood granu- kg midazolam (Rotexmedica GmbH, Trittau, late bedding was used (Lignocel, Altromin, Lage, Germany). As soon as the toe pinch reflex had dis- Germany). Autoclaved commercial pellet diet (Altromin appeared, 1 mg/kg meloxicam (MetacamÕ, 2 mg/ml, 1314) and autoclaved water were provided ad libitum. Boehringer Ingelheim) for pain relief and 0.05 mg/kg Garrels et al. 367

Atropin (Atropinsulfat, 0.5 mg/ml, B. Braun) to prevent 48 h later of 5 IU human chorionic gonadotropin bradycardia were injected subcutaneously (s.c.). The eyes (hCG, Primogonyl-1000, Schering Pharma, Berlin, were covered with eye ointment (Bepanthen, Bayer, Germany) was administered. One or two batches with Leverkusen, Germany) to protect the cornea. Post-sur- about 20 hybrid embryos were frozen as control with gery the cages were placed on a warming plate (25–30C) every freezing cycle. Routinely, controls are stored for until full recovery of the animals. at least one month in liquid nitrogen after freezing, before they are thawed and transferred to embryo Vasectomy transfer recipients to conﬁrm a successful archiving.

CD2F1 males were vasectomized by cauterization of both Experimental design ductus deferentes, as described in detail elsewhere.8,9 Briefly, the anaesthetized male was placed in a dorsal Vasectomized CD2F1 males and Gapdhs-/- males were body position and the abdomen was opened by an inci- monogamously mated in parallel overnight to sion at the linea alba 0.5 cm cranial from the pelvis. The B6CBAF1 females on seven independent experimental fat pad surrounding the right testes was grasped with days (18–20 matings per group and experimental day). forceps and pulled out until the ductus deferens was Females in pro-oestrus or oestrus mated during the visible. A small piece of the spermatic duct (0.3 cm) night. The following morning females were checked was removed by cauterization (Small Vessel Cauterizer, for the presence of a vaginal plug (VP) to determine Fine Science Tools GmbH, Heidelberg, Germany). The the ‘plug rate’. At the same day we transferred rando- procedure was then contralateral repeated. The testicles mized 6–14 frozen-thawed control embryos per female were shifted back and the abdominal cavity was closed to determine the ‘pregnancy rate’ and the portion of in two layers with sutures. Two to three weeks later the embryos that developed to term (‘birth rate’). males were test mated to ensure their sterility before using them for mating to induce pseudopregnancy. Breeding efforts to generate vasectomized males Embryo transfer The colony index (CI) was used to determine the For the induction of pseudopregnancy B6CBAF1 number of required mating to generate a given females were mated overnight either with Gapdhs-/- or number of Gapdhs-/- and CD2F1 males. The CI was surgical vasectomized CD2F1 males. The males were calculated by dividing the total number of offspring by used once a week. Vaginal plug positive mice were the number of mated females per week.10 Working time used for unilateral embryo transfer, performed as for vasectomy (including peri- and postoperative care) described in detail elsewhere.9 Briefly, after anaesthesia and genotyping were evaluated and compared. females were placed in a ventral body position. A longitudinally incision in the lumbar area was made. Next, Statistics the abdominal cavity was opened and the ovary with oviduct was carefully pulled forward. The bursa ovarica We used www.biomath.info/power/PowerAnalysis. was opened and two-cell embryos were transferred into html to calculate the most appropriate group size. the oviduct. The reproductive tract was gently placed Results were analysed using the GraphPad Prism5 pro- back into the abdominal cavity, the peritoneum sutured gram. Plug rate and pregnancy rate were tested with and the skin closed with wound clamps (Michel suture chi square statistics (Fisher’s exact test). With the clips 7.5 1.75 mm, Aesculap, Tuttlingen, Germany). assumption of Gaussian distribution for the number Birth was expected approximately 19–20 days later. of transferred embryos developed to term, we performed two-tailed t-tests to determine statistical differ- -/- Embryos ences. The colony indices of Gapdhs and CD2F1 were compared by one-tailed t-tests. Differences of the To avoid extra embryo production, we transferred means with p < 0.05 were judged as significant. frozen-thawed two-cell ‘control embryos’ from our institutional embryo-freezing programme. For this purpose hybrid two-cell embryos were collected at day 2 Results post coitus from superovulated BALB/cJHanZtm Plug rate females mated with DBA/2JHanZtm males. For super- ovulation an i.p. injection of 5 IU pregnant mare’s After overnight mating of 132 females with vasecto- serum gonadotropin (PMSG, Intergonan, Vemie mized CD2F1 males, 28 (21.2 %) had a vaginal plug Veterina¨ r Chemie GmbH, Kempen, Germany) and next morning. In the Gapdhs-/- group 158 females were 368 Laboratory Animals 52(4) mated overnight. Next morning 39 (24.6 %) females The difference in the mean number of transferred were identified as vaginal plug positive. The two-sided embryos per recipient between both mating groups Fisher’s exact test (with a < 0.05) showed no significant was not significant (8.5 0.4 for Gapdhs-/- and difference (Table 1). 7.4 0.3 for vasectomized males; p ¼ 0.45) and a pos- Females with a vaginal plug were judged as ‘pseudo- sible impact on the presented results can be excluded. pregnant’ and according to the number of thawed embryos the necessary number of embryo transfer Comparison of breeding efforts to generate recipients per experimental day was selected (day 0.5 vasectomized males pseudopregnancy was additionally confirmed during embryo transfer by the presence of a swollen ampulla). For routine embryo transfers, a group of 40 vasectomized males was permanently available in the Central Pregnancy rate Animal Facility of the Hannover Medical School. The following calculations refer therefore to the number of Embryo transfers were conducted on five independent 40 vasectomized males. experimental days. Fourteen of 17 (82.3%) pseudopregnant females previously mated to vasectomized CD2F1 Colony index males became pregnant after embryo transfer. Simultaneously, 16 (80.0 %) of 20 females plug positive The reproductive performance of a strain is represented after mating to Gapdhs-/- males became pregnant and by the colony index (CI) and can be used to determine gave birth. The two-sided Fisher’s exact test (with the number of required breeding females to generate a a < 0.05) showed no significant difference (Table 1). given number of mice. With the assumption of a sex ratio of approximately Embryo development 1:1 we need 80 pups to produce 40 CD2F1 males. In contrast, only 25 % of the male offspring were Sixty of 125 two-cell embryos transferred to 17 recipi- homozygous for the Gapdhs knockout mutation, due ents developed to term. This results in a frequency of to the use of heterozygous breeding males. Therefore, born offspring of 48.04 6.7% for pseudopregnant 160 pups were necessary to produce 40 males with the recipients produced by mating with vasectomized appropriate sterile phenotype. CD2F1 males. An almost identical frequency of The CI based on live born pups was 2.69 0.27 for 48.7 7.2% was observed for females mated to the Gapdhs line (20 breeding females analysed) and Gapdhs-/- males. In total 169 embryos were used in 2.25 1.5 for CD2F1 (31 breeding females analysed). for 20 transfers. One recipient of this group which The differences between the CIs were tested as not sig- received seven embryos was excluded from the study nificant. The resulting sex ratio among offspring of because of infanticide before the number of pups both strains was as expected approximately 1:1. The could be recorded. Altogether, 79 of 162 transferred number of litters necessary to produce homozygous embryos developed to term in recipients mated with Gapdhs-/- males was calculated assuming a Mendelian Gapdhs-/- males (Table 1). inheritance of the knockout allele. Meaning 60 litters are needed to produce 40 Gapdhs-/- males and 36 litters to provide 40 CD2F1 males for vasectomy (Table 2).

Table 1. Plug rate, pregnancy rate and frequency of born Expenditure of time and severity offspring in the experimental groups. classification of animal treatments -/- Vasectomized Gapdhs Expenditure of time for vasectomy, i.e. duration of sur- CD2F1-males males gery, postoperative care over the next days (control of Plug rate in % (n) 21.2 (28/132) 24.6 (39/158) movement, food- and water uptake, wound healing Pregnancy rate in % (n) 82.3 (14/17) 80.0 (16/20) etc.) was calculated to be 60 minutes per male. Taking of an ear biopsy, followed by PCR analysis for geno- Born offspring in % of 48.0 6.7 48.7 7.3 transferred (60/126) (79/162) typing, takes about 30 minutes per male. embryos (n) Severity classification of the treatments was made according to Directive 2010/63EU, Annex VIII. Males were mated to B6CBAF1 females. Results were determined Embryo transfer had to be classified as ‘moderate’ for every single experimental day. For plug and pregnancy rate a Fisher’s exact test was performed. To compare the rate of born with less than seven days of postoperative pain. offspring a t-test was performed (results are given as mean SD). A licence for this procedure was obligatory. Ear All differences were not significant. biopsy was classified as ‘mild’ and postoperative pain Garrels et al. 369

Table 2. Expenditure to produce 40 vasectomized or more surplus animals compared to the production of mutant males. F1-hybrid males for vasectomy. However, a male with hybrid or outbred background can be used for mating CD2F1 Gapdhs-/- for several months and therefore a large-scale produc- Colony index 2.25 1.5 2.69 0.27 tion of animals to produce sterile males is unnecessary Number of pups to be 80 160 for both options. produced Vasectomy, postoperative care and test breeding for Number of litters to be 36 60 F1-hybrids were more time-consuming than the neces- produced sary genotyping of mutants, including the collection of Litter size* 8.1 0.5 10.7 0.1 tissue samples, DNA extraction and PCR reaction. Gender distribution (,/<) 1.37 0.14 1.03 0.09 However, the expenditure of time for surgery was Genotype distribution n.a. 1.7 0.2 balanced by the need for more breeding cages and asso- (Gapdhsþ/-/Gapdhs-/-) ciated time for animal care for the Gapdhs line. Obvious differences between vasectomy and genetic- *The difference between the average litter size of CD2F1 and ally induced infertility exist in the assessment of the Gapdhs mice was significant; two out of 64 litters analysed did not produce Gapdhs-/- mice; n.a., not applicable. degree of severity. According to the Directive 2010/ 63EU, Annex VIII, a vasectomy in mice needs to be licensed by the competent authority and only author- ized and appropriately qualified persons are allowed was considered for less than one day. No licence was to do it. necessary because the primary purpose of ear punching Besides sterility, the Gapdhstm1Dao males showed no was the identification of the animal. other changes in their phenotype. Vasectomy, however, is carried out as surgery performed under general Discussion anaesthesia associated with post-surgical pain, scored as ‘moderate’ severity and required appropriate anal- In the present study we assessed (i) whether sterile gesia. Metacam injection was applied already during males generated by a genetic modification can be reli- anaesthesia and gave relief of post-surgical pain for ably used to induce pseudopregnancy in females like up to 24 h. Vasectomized males recovered well during commonly used vasectomized males, (ii) whether the the observation period without any evidence of abnor- use of these mutant males or surgically vasectomized mal behaviour which would indicate strong and lasting males is more efficient and (iii) which method should be pain for the animals. Furthermore, we never found preferred in the view of animal welfare aspects and the indications for wound infections. Long term effects of 3R principle. vasectomy in mice have been described.13 The effects, The results did not reveal any significant differences however, were of histological nature and did not seem in plug rate or pregnancy rate between females that to influence wellbeing of the animals. We routinely use were mated to vasectomized males or sterile Gapdhs vasectomized hybrid males for up to 18 months without mutants. Also the portion of transferred embryos that any health problems that could be assigned to the sur- developed to term (birth rate) did not differ. gery. The length of useful life span of Gapdhs mutants Furthermore, males of both experimental groups for efficient induction of pseudopregnancy has still to showed a good plug forming ability for easy mating be evaluated. control. This is an important finding because the copu- In conclusion: For the induction of pseudopreg- latory plug in mice appears to be important for nid- nancy in female mice genetically modified Gapdhs-/- ation and maintaining of pregnancy. Recently, it was males are as suitable, with the same efficiency, as com- shown that despite sufficient oocyte fertilization fewer monly used vasectomized hybrid males. The need to females gave birth when the plug forming ability of the produce more offspring to provide homozygous male was disrupted.11 An impact of the genetic back- mutant males was the only disadvantage of the ground on characteristics of the vaginal plug has also method, compared to surgically vasectomized CD1F1 been reported.12 males. This disadvantage, however, was matched by Importantly, no leakiness in fertility was found for animal welfare aspects. Moreover, surplus animals homozygous Gapdhs knockout males, making test with a wrong genotype or sex, produced by mating breeding, as commonly applied for vasectomized Gapdhs-/- females with Gapdhsþ/- males could be males, dispensable. In contrast, nearly double breeding used for other purposes (ex. as sentinel animals in effort was necessary to provide infertile male mutants. microbiological monitoring programs). Table 3 directly Only 25% of the offspring are homozygous knock- compares the evaluated parameters in order to compare out males with the desired phenotype, resulting in both approaches. 370 Laboratory Animals 52(4)

Table 3. Summary of the evaluated parameters.

Vasectomized Criteria Gapdhs-/- CD2F1 Comment

Plug rate 0 0 Ability to mate with a female successfully, as indicated by the presence of a vaginal plug after mating. We did not find any statistical difference between homozygous Gapdhs-/- males and surgically vasectomized CD2F1 males. Pregnancy rate 0 0 Portion of females that gave birth after successful mating with sterile males and transfer of cryo-thawed embryos. We did not find any statistical difference between homozygous Gapdhs-/- males and surgically vasectomized CD2F1 males. Frequency of 0 0 Portion of transferred cryo-thawed embryos that developed born offspring to term after transfer to successfully mated females. We did not find any statistical difference between homozygous Gapdhs-/- males and surgically vasectomized CD2F1 males. Time exposure þ - Less time was needed to take biopsies from Gapdhstm1Dao mice for genotyping compared to the time needed for vasectomy of the CD2F1 males (including perioperative care). Breeding effort - þ Despite the same colony-index, more litters were needed to produce a given number of homozygous Gapdhs-/- males compared to the same number of CD2F1 males. More surplus mice (i.e. mice with a ‘wrong’ genotype) were produced in the Gapdhs-/- colony. expenses þ - Financial costs for genotyping Gapdhstm1Dao mice were less than vasectomy of CD2F1 males. Personnel expenditures* þ - The qualification level of persons that perform surgery (at least a veterinarian is necessary for anaesthesia) had to be higher than those who take biopsies for genotyping (animal care takers can take biopsies). Welfare impact þ - Severity for vasectomy had to be classified as ‘moderate’, whereas taking of biopsies for genotyping were classified as ‘mild’.

‘0’ no differences were found, ‘þ’ more advantageous, ‘-’ less advantageous if genetically vasectomized Gapdhstm1Dao males were compared to surgically vasectomized CD2F1 males. *Personnel expenditures based on the working document on the development of a common education and training framework to fulfil the requirements under the Directive 2010/63/EU of the Expert Working Group.15

Especially for the production of germ free mice in of produced surplus animals can reasonably be used for isolators, the use of sterile Gapdhs males might be a other purposes. good alternative to vasectomy because surgical intervention under these conditions are laborious and Acknowledgements bears the risk for unwanted hygienic contamination. In combination with non-surgical (transcervical) Gapdhs knockout mice were produced in E.M. Eddy’s lab at embryo transfer,14 the use of sterile Gapdhs males the National Institute of Environmental Health Sciences, National Institutes of Health and characterized in the lab of would enable research groups, without speciﬁc equip- D O’Brein at UNC. ment or appropriate persons for surgical interventions, to apply several reproductive techniques which otherwise would require surgical intervention. From the Declaration of Conflicting Interests animal welfare aspect, genetically sterile males should The author(s) declared no potential conﬂicts of interest with be the preferred alternative for the induction of pseu- respect to the research, authorship, and/or publication of this dopregnant females, provided that the higher number article. Garrels et al. 371

Funding of guidelines for health monitoring of rodents and rabbits. Lab Anim 2014; 48: 178–192. The author(s) received no ﬁnancial support for the research, 8. Hogan BB, Costantini F and Lacy E. Manipulating the authorship, and/or publication of this article. mouse embryo, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1994. 9. Dorsch M. Cryopreservation of preimplantation embryos References and gametes, and associated methods. In: Hedrich HJ 1. Lipkow J. Die Bedeutung des Vaginalpfropfes bei der (ed.) The laboratory mouse, 2nd ed. Amsterdam: weißen Maus (Mus-Musculus L). Naturwissenschaften Elsevier, 2012, Chapter 4.7. 1959; 46: 93. 10. Wedekind D, Reifenberg K and Hedrich HJ. Genetic 2. Sutter A and Lindholm AK. The copulatory plug delays monitoring of inbred strains of mice. In: Hedrich HJ ejaculation by rival males and affects sperm competition (ed.) The laboratory mouse, 2nd ed. Amsterdam: outcome in house mice. J Evol Biol 2016; 29: 1617–1630. Elsevier, 2012, Chapter 4.5. 3. Yang JJ, Larsen CM, Grattan DR, et al. Mating-induced 11. Dean MD. Genetic disruption of the copulatory plug in neuroendocrine responses during pseudopregnancy in the mice leads to severely reduced fertility. PLoS Genet 2013; female mouse. J Neuroendocrinol 2009; 21: 30–39. 9: e1003185. 4. Miki K, Qu W, Goulding EH, et al. Glyceraldehyde 3- 12. Mangels R, Young B, Keeble S, et al. Genetic and pheno- phosphate dehydrogenase-S, a sperm-specific glycolytic typic influences on copulatory plug survival in mice. enzyme, is required for sperm motility and male fertility. Heredity 2015; 115: 496–502. Proc Natl Acad Sci USA 2004; 101: 16501–16506. 13. Singh SK and Chakravarty S. Histologic changes in the 5. Brunner S, Colman D, Travis AJ, et al. Overexpression of mouse testis after bilateral vasectomy. Asian J Androl RPGR leads to male infertility in mice due to defects in 2000; 2: 115–120. flagellar assembly. Biol Reprod 2008; 79: 608–617. 14. Cui L, Zhang Z, Sun F, et al. Transcervical embryo trans- 6. Haueter S, Kawasumi M, Asner I, et al. Genetic fer in mice. J Am Assoc Lab Anim Sci 2014; 53: 228–231. vasectomy-overexpression of Prm1-EGFP fusion protein 15. Expert Working Group (EWG) on Education and in elongating spermatids causes dominant male sterility in Training. National Competent Authorities for the imple- mice. Genesis 2010; 48: 151–160. mentation of Directive 2010/63/EU on the protection of 7. Ma¨ hler M, Berard M, Feinstein R, et al. FELASA rec- animals used for scientific purposes, http://ec.europa.eu/ ommendations for the health monitoring of mouse, rat, environment/chemicals/lab_animals/pdf/Endorsed_E-T. hamster, guinea pig and rabbit colonies in breeding and pdf (2014, accessed 19–20 February). experimental units. FELASA working group on revision

Re´sume´ La souris de laboratoire est le mode`le animal le plus utilise´ en recherche biome´dicale. Plusieurs techniques de reproduction artificielle, telles que la revitalisation de souches cryopre´serveés, la rede´rivation apre`s contamination hygieńique et la production de mode`les murins transgeńiques, exigent le transfert d’embryons preímplantatoires dans des femelles porteuses. Les grossesses nerveuses sont essentielles chez les femelles et sont induites par l’accouplement avec des maˆles ste´riles. Des maˆles vasectomise´s chirurgicale- ment sont geńe´ralement utilise´sa` cette fin. Comme alternative, des souches de souris geńe´tiquement modifieés ont e´te´ identifieés, dans lesquels des maˆles homozygotes infertiles sont sexuellement actifs. Ici, nous avons e´tudie´ la pertinence des maˆles Gapdhstm1Dao geńe´tiquement ste´rilise´s en conditions de laboratoire, en ce qui concerne les taux de prise, les taux de grossesse et la fre´quence des petits ne´s apre`s transfert d’embryons. Nos re´sultats n’ont montre´ aucune diffe´rence significative concernant ces aspects entre les maˆles Gapdhs1tm Dao et et les maˆles CD2F1 vasectomise´s. Nous avons en outre e´value´ les efforts visant a` obtenir un nombre de´fini de maˆles ste´riles, soit par se´lection de mutants ste´riles soit par vasectomie chirurgicale, et examine´ l’incidence de ces deux options sur le bien-eˆtre animal. En conclusion, les maˆles ste´riles de la ligne Gapdhstm1Dao constituent une alternative fiable aux maˆles vasectomise´s pour l’induction de grossesses nerveuses, et peut contribuer au raffinement de la proce´dure en e´vitant une intervention chirurgicale.

Abstract Die Labormaus ist das meistverwendete Tiermodell in der biomedizinischen Forschung. Verschiedene ku¨nstliche Reproduktionstechniken, wie die Revitalisierung von kryokonservierten Sta¨mmen, die Reaktivierung nach hygienisch bedingten Kontaminationen und die Herstellung transgener Mausmodelle, erfordern die U¨bertragung von Pra¨implantationsembryonen auf Leihmu¨tter. Pseudoschwangerschaft ist bei Empfa¨ngerinnen unerla¨sslich und wird durch Paarung mit sterilen Ma¨nnchen induziert. Ha¨ufig werden 372 Laboratory Animals 52(4) zu diesem Zweck chirurgisch vasektomierte Ma¨nnchen eingesetzt. Als Alternative wurden gentechnisch vera¨nderte Maussta¨mme identifiziert, bei denen homozygote unfruchtbare Ma¨nnchen sexuell aktiv sind. Hier untersuchten wir die Eignung genetisch unfruchtbarer Gapdhstm1Dao-Ma¨nnchen unter routinema¨ßigen Laborbedingungen im Hinblick auf Pfropfraten, Tra¨chtigkeitsraten und Frequenz der Nachkommen nach dem Embryotransfer. Unsere Ergebnisse zeigten fu¨r diese Aspekte keine signifikanten Unterschiede zwischen Gapdhstm1Dao- und vasektomierten CD2F1-Ma¨nnchen. Daru¨ber hinaus evaluierten wir die Bemu¨hungen, eine definierte Anzahl steriler Ma¨nnchen entweder durch Zu¨chtung steriler Mutanten oder chirurgische Vasektomie zu erhalten, und befassten uns mit den Auswirkungen beider Optionen auf den Tierschutz. Zusammenfassend la¨sst sich sagen, dass unfruchtbare Ma¨nnchen der Gapdhstm1Dao-Linie eine zuverla¨ssige Alternative zu vasektomierten Ma¨nnchen fu¨r die Induzierung der Pseudoschwangerschaft darstellen und zur Verbesserung des Verfahrens aufgrund des Wegfalls chirurgische Eingriffe beitragen ko¨nnen.

Resumen El ratoń de laboratorio es el modelo animal ma´s utilizado en la investigacioń biome´dica. Varias tećnicas de reproduccioń artificial, como la revitalizacioń de cepas crioconservadas, la rederivacioń tras contaminaciones higieńicas y la produccioń de modelos de ratoń transgeńicos, requieren la transferencia de embriones de preimplantacioń a madres subrogadas. El pseudoembarazo es esencial en las hembras receptoras y se induce mediante el apareamiento con machos este´riles. Por lo general, suelen utilizarse machos con vasectomıá para este fin. Como alternativa, las cepas de ratoń gene´ticamente modificadas han sido identi- ficadas, en las que los machos este´riles homocigotos son sexualmente activos. En este estudio hemos investigado la idoneidad de los machos Gapdhstm1Dao gene´ticamente este´riles en unas condiciones rutinarias de laboratorio respecto a tasas de taponamiento, tasas de embarazo y la frecuencia de nacimiento de crıás tras la transferencia de embriones. Nuestros resultados no han mostrado unas diferencias significativas para estos aspectos entre los machos Gapdhstm1Dao y los CD2F1 con vasectomıá. Asimismo, hemos evaluado los esfuerzos necesarios para obtener un nu´mero definido de machos este´riles mediante la crıá de mutantes este´riles o vasectomıá quiru´rgica y, asimismo, hemos evaluado el impacto de ambas opciones sobre el bienestar animal. En conclusioń, los machos este´riles de la lıńea Gapdhstm1Dao son una alternativa fiable a los machos con vasectomıá para la induccioń de pseudoembarazo y pueden contribuir al refinamiento del procedimiento evitando la intervencioń quiru´rgica. laboratory an imals Original Article limited Laboratory Animals 2018, Vol. 52(4) 373–383 ! The Author(s) 2018 Softened food reduces weight loss Reprints and permissions: sagepub.co.uk/ in the streptozotocin-induced male journalsPermissions.nav DOI: 10.1177/0023677217747915 mouse model of diabetic nephropathy journals.sagepub.com/home/lan

Sisse A Nørgaard1,2,3, Fredrik W Sand1, Dorte B Sørensen2, Klas SP Abelson4 and Henrik Søndergaard3

Abstract The streptozotocin (STZ)-induced diabetic mouse is a widely used model of diabetes and diabetic nephropathy (DN). However, it is a well-known issue that this model is challenged by high weight loss, which despite supportive measures often results in high euthanization rates. To overcome these issues, we hypothesized that supplementing STZ-induced diabetic mice with water-softened chow in addition to normal chow would reduce weight loss, lower the need for supportive treatment, and reduce the number of mice reaching the humane endpoint of 20% weight loss. In a 15 week STZ-induced DN study we demonstrated that diabetic male mice receiving softened chow had reduced acute weight loss following STZ treatment (p ¼ 0.045) and additionally fewer mice were euthanized due to weight loss. By supplementing the diabetic mice with softened chow, no mice reached 20% weight loss whereas 37.5% of the mice without this supplement reached this humane endpoint (p ¼ 0.0027). Excretion of corticosterone metabolites in faeces was reduced in diabetic mice on softened chow (p ¼ 0.0007), suggesting lower levels of general stress. Finally, it was demonstrated that the water-softened chow supplement did not significantly affect the induction of key disease parameters, i.e. %HbA1C and albuminuria nor result in abnormal teeth wear. In conclusion, supplementation of softened food is refining the STZ-induced diabetic mouse model significantly by reducing stress, weight loss and the number of animals sacrificed due to humane endpoints, while maintaining the key phenotypes of diabetes and nephropathy.

Keywords streptozotocin-induced diabetes, diabetic nephropathy, weight loss, refinement, reduction

Date received: 10 July 2017; accepted: 11 November 2017

Introduction resembling the early pathological changes in human The streptozotocin (STZ)-induced diabetic mouse has for DN manifested as albuminuria and glomerular mesan- many years been one of the most used models in both gial expansion.9 diabetes and diabetic nephropathy (DN) research.1,2 STZ, which was originally discovered as an anti- 1Diabetes & Cardiovascular Pharmacology, Novo Nordisk A/S, biotic,3 was further developed in oncology as a che- Denmark 2 motherapeutic agent against pancreatic islet cell Department of Veterinary Disease Biology, University of 4 Copenhagen, Denmark cancer. Since the discovery of the diabetogenic eﬀect 3 5 Diabetes Complications Pharmacology, Novo Nordisk A/S, of STZ in the 1960s, the STZ-induced diabetes model Denmark has been a widely used rodent model in both diabetes 4Department of Experimental Medicine, University of Copenhagen, and DN research.6 STZ is a glucose analogue which is Denmark transported into pancreatic beta cells via GLUT2 glu- Corresponding author: cose transporters where it accumulates and triggers 7,8 Sisse A Nørgaard, Diabetes & Cardiovascular Pharmacology, Novo necrosis. The STZ-induced hyperglycaemia eventu- Nordisk A/S, Novo Nordisk Park 1, 2760 Ma˚løv, Denmark. ally induces the development of kidney damage Email: [email protected] 374 Laboratory Animals 52(4)

STZ-induced diabetes is typically induced in male potentially improve the quality of the outcome should mice and rats, since males are more sensitive to STZ be pursued. than females.10–13 The susceptibility for STZ in mice is The aim of this study was to investigate if supple- also affected by the mouse strain due to differences in menting STZ-induced diabetic male mice with water- GLUT2 expression.12 softened chow in addition to their regular chow pellets The main advantage of the STZ-induced diabetes could result in (1) reduced acute weight loss, (2) model is the cost effectiveness and the simple technical reduced euthanization rate due to humane endpoints, requirements needed to induce stable diabetes and signs (3) lower need for supportive treatment e.g. insulin of nephropathy in mice. However, despite its extensive injections, and (4) reduced stress level in the mice. use, STZ-induced diabetes is variable and no generally Finally, it was investigated if the water-softened chow accepted standardized protocol exists regarding mouse supplement would significantly affect the induction of strain, STZ administration, health monitoring or sup- diabetes and DN. portive treatment. One of the main issues with STZ-induced diabetes in mice is that the mice often suffer significant and acute weight loss resulting in Materials and methods 9,14–16 high euthanization rates. This weight loss is Ethics statement likely to be the result of the acute onset and progression of diabetes, but the acute weight loss might also be The study was approved by The Animal Experiments caused both by the repeated fasting of the animals Inspectorate, Ministry of Environment and Food of and the acute toxicity of STZ.14,17 In addition to Denmark (license no. 2014/15-0201-00429). Any mice being toxic to beta-cells, STZ has been found to have in this experiment that showed signs of compromised a direct toxic effect especially on the kidneys and health (inadequate activity/moribund) or had lost more other organs such as the liver and intestines.18,20–22 than 20% body weight was terminated immediately Originally, a single high dose of STZ (in mice according to the EU Directive 2010/63/EU on the pro- 150–200 mg/kg) was used to induce diabetes. With tection of animals used for scientific purposes. this high dose, the STZ induces a direct cytotoxic effect on the b-cells, leading to fast induction of Mice severe diabetes with undetectable levels of insulin.23 But the toxic effect on especially the kidney is also Fifty-five 129S2/SvPasCrl male mice (6–8 weeks old, more pronounced with this high dose of STZ.9,19 min. 21 g) were purchased from Charles River, To overcome the acute toxicity of STZ, the Diabetic Germany. The mice were housed in groups of 6–10 Complications Consortium (DiaComp) recommends mice in type IV open cages (595 mm 380 mm multiple low doses of STZ, 5 50 mg/kg given on 200 mm) from Scanbur, Karlslunde, Denmark, located five consecutive days,24,25 resulting in a slower progres- in cabinets (NOVOtainer, Scanbur, Karlslunde, sion of hyperglycaemia driven by apoptosis of the Denmark) with up to 16 cages in each. The mice had b-cells.26,27 In this study, we use a protocol which we free access to tap water (from the Scantainer’s watering refer to as double intermediate dose, where we inject system as well as extra water bottles in cage lid) and to the mice with 100 mg/kg STZ on day 1 and 4, as pre- standard mouse chow pellets (Altromin 1324; viously published by Meier et al.28 We have found this Brogaarden ApS, Lynge, Denmark). Furthermore, as protocol to have the same consistency in induction of enrichment they were given paper nesting material, diabetes and similar or less loss in body weight com- wooden blocks, a cardboard tube (31008, 30968, pared with 5 50 mg/kg (unpublished data) and more- 31005, Brogaarden ApS, Lynge, Denmark), a plastic over this protocol has the advantage that the animals hide (special order, Bach Ventilation ApS, Søborg, are only fasted and injected with STZ twice. Denmark), and a hemp rope (4101022006, Fyns Kran The high weight loss of the STZ-treated mice affects Udstyr A/S, Vallensbæk Strand, Denmark) hanging the overall animal welfare and requires larger group from the cage lid. The mice were kept in a room with sizes to ensure appropriate power and extensive 25C, 30–60% relative humidity, and a 12 h day/night health monitoring throughout the study period. cycle. Ear punches were used as an identification However, many reports do not account for weight method. Before study start the mice were acclimatized loss and the loss of animals making the extent of this for 2 weeks. problem difficult to determine. It is nevertheless certain that there is a need for refining this animal model so Experimental design fewer mice reach significant weight loss. Thus, initia- tives to increase the animal welfare, reduce the The mice were allocated to either STZ or vehicle groups number of animals required in studies as well as and further divided into groups receiving either normal Nørgaard et al. 375 chow pellets or both chow pellets and water-softened non-pelleted chow and enough tap water to moisten non-pelleted chow (Altromin 1324, Brogaarden ApS, all the food (around 35 mL). Lynge, Denmark). The animals were weighed three times a week. If an At study week 6 the mice receiving the softened food animal had lost more than 10% body weight compared (both non-diabetic and diabetic) were further divided to start weight, 1 mL 0.9% saline was administered into two groups, being housed either with or without intraperitoneally (i.p.) once a day. In addition, Lantus red plastic igloos (Mouse Igloo, K3327, Bio-Serv, (insulin glargine, commercially available), was admin- Flemington, USA) during the metabolic cage housing istered at 6 U/kg using a NovoPen Echo. This support- described below. The rationale for this was to test if ive treatment was given three times a week for at least a giving the mice a hide in the metabolic cages could whole week and until the weight loss was no longer reduce weight loss or the stress hormone response exceeding 10%. seen when housing mice in metabolic cages. Since we Any animal that experienced more than 20% weight found no difference for either of these readouts, data loss was euthanized immediately. from the two groups was merged in all analysis and this The mice were euthanized under isoflurane anaesthesia test was not repeated at week 14 (see Supplementary by intra-cardiac perfusion with 0.9% NaCl with heparin. Figures 1 and 2 online). All the mice only receiving pellets were given an igloo. STZ administration The group sizes for the merged groups were at the beginning of study then as follows: vehicle, pellets: STZ (S-0130, Sigma-Aldrich, St Louis, MO, USA) was n ¼ 7; vehicle, softened food: n ¼ 12; STZ, pellets: reconstituted in a sodium citrate buffer (pH 4.5, n ¼ 16; STZ, softened food: n ¼ 20. The group sizes 1.06448, Merck, Billerica, USA) in a concentration of were chosen based on previous studies and experience. 10 mg/mL. Immediately after reconstituting the STZ, Since loss of animals in the diabetic groups was the mice (8–10 weeks of age, >22 g) were injected i.p. expected more animals were assigned to the STZ with either 100 mg/kg STZ or 10 mL/kg sodium citrate groups. Furthermore, the groups receiving softened buffer on study day 1 and 4 after being fasted for 4 h food had more animals to make it possible to test the (from 7 to 11 a.m.). hypothesis of mouse igloo lowering stress in the metabolic cages. Monitoring diabetes and DN The softened food was given once every morning in the bottom of the cage in a 100 mL plastic Mice were weighed three times per week to monitor weighing boat (HEA1424C, Heathrow Scientific, their body weight and general well-being. Blood glucose Vernon Hills, USA) from the day of the first STZ injec- was measured two times per week and %HbA1c every tion. A portion contained approximately 25 g of third week throughout the study to investigate the

(a) (b) 30 200 * STZ STZ

20 100

10 0 Vehicle, pellets Vehicle, softened food 0 -100 STZ, pellets AUC (g x day) -10 STZ, softened food -200 Body weight change (%)

-20 -300 0 5 10 15 20 25 STZ, STZ, pellets softened food Time (day)

Figure 1. Water-softened chow reduces acute weight loss following STZ injection. (a) Percent change in body weight from day 1 (prior to STZ injection) until the first occurrence of a euthanization at day 22. The mice were injected with either 100 mg/kg STZ or vehicle at day 1 and 4, marked by vertical dotted lines in the figure. Data are shown as mean SD. (b) Area under the curve for the body weight change of individual mice in the two STZ groups. *p ¼ 0.045, by two-tailed students t-test. Data are shown as individual points as well as mean SD. 376 Laboratory Animals 52(4)

(a) (b)

Figure 2. Water-softened chow reduced frequency of supportive treatment and humane endpoints. (a) Table showing the frequency of days with supportive insulin and saline injections given to the diabetic mice when reaching 10% weight loss of total possible monitoring days. ****p < 0.0001, RR ¼ 2.45 (CI: 1.928–3.108), by two-tailed Fisher’s exact test. (b) Graph showing the total number of mice euthanized during the study due to 20% weight loss. No mice were euthanized in the softened chow group due to 20% weight-loss. **p ¼ 0.0027, by log-rank test. effect of the softened food on diabetes induction and Quantification of corticosterone metabolites development. Blood samples for BG measurements in faecal samples were obtained from non-fasted, conscious mice by puncturing the tail vein with a lancet. 5 mL blood was The faecal samples were kept at 20C until analysed. collected into heparinized capillary tubes and trans- The corticosterone metabolites were quantified using a ferred to 250 mL glucose buffer (0201-0002-024, EKF protocol modified from a previous description.29 The diagnostics, Cardiff, UK). Blood glucose concentration faecal samples were added to 96% ethanol (3 mL/g was measured on a Biosen 5040 (EKF Diagnostics, faeces) and placed on a tipping table overnight. After Cardiff, UK). centrifuging twice in 20 min at 4000 r/min, 200 mL of the Blood (5 mL) for %HbA1c measurements was col- supernatants were evaporated to dryness and the resi- lected into heparinized capillary tubes from the tail dues dissolved in 200 mL PBS. The samples were then vein from non-fasted, conscious mice, transferred to analysed using a corticosterone ELISA (EIA-4164; 500 mL HbA1c assay buffer (11488457, Roche DRG Diagnostics GmbH, Marburg, Germany). Diagnostics GmbH, Mannheim, Germany), frozen at 20 C until analysis and analysed on a Cobas 6000 Statistical analysis (Roche Diagnostics Ltd, Rotkreuz, Switzerland). At study week 6 and 14 the mice were placed indi- Statistical tests were done using GraphPad Prism soft- vidually in metabolic cages (Techniplast, Buguggiate, ware. The specific statistical tests used for each data set Italy) for 19 h where urine was collected for albumin- are mentioned in the respective figure legend. A p-value uria measurements and faeces for corticosterone meas- of 0.05 was considered statistically significant. All mice urement (only week 6). When the mice were taken out, were included in every analysis but since several the urine was weighed and samples for albumin meas- mice were lost during the study, data from these mice urements were diluted 20 in 50 mM TBS pH 8.0, 1% are missing in some of the analysis. BSA, 0.05% Tween20 and stored at 20C until analysis. Albumin concentration was measured using an assay based on an ELISA kit from Bethyl Results Laboratories Inc. (E90-134, Bethyl Laboratories, Inc., Weight loss, supportive treatment and Montgomery, USA). Calibrator and controls was made humane endpoints using albumin from Sigma-Aldrich (A3139-10 mg, Sigma-Aldrich, St Louis, MO, USA). Coating buffer, The water-softened chow supplement was initiated wash solution and blocking buffer was additionally from the first injection of STZ and soon after introduc- purchased at Sigma-Aldrich (C3041, T9039, T6789, tion the mice were observed to eat the softened food in Sigma-Aldrich, St Louis, MO, USA). The 19 h sample addition to their regular chow pellets. The weight of the was used to calculate the 24 h urinary albumin excre- mice was monitored throughout the study. During the tion rate. first three weeks, no mice were euthanized from any of Nørgaard et al. 377 the four groups. Thus, to evaluate the effect of the Any mouse experiencing weight loss of 10% or more water-softened chow on acute weight loss following received supportive treatment with insulin and saline STZ injection, mean body weight change for the first according to protocol (see Materials and methods). 3 weeks is shown in Figure 1(a). Compared to the vehi- The STZ-induced diabetic animals given water-softened cle groups, which gained weight, both STZ-injected chow in addition to normal chow experienced a signifi- groups lost weight as expected. However, when com- cantly lower frequency of days with supportive injec- paring the AUC of the weight change curve for the two tions of insulin and saline per total number of STZ groups (Figure 1(b)), the STZ mice given water- monitoring days (Figure 2(a); p < 0.0001). softened chow in addition to chow pellets lost signifi- Any animal experiencing more than 20% weight loss cantly less weight (p ¼ 0.045) during the first 3 weeks. compared to start weight was euthanized immediately Beyond the first three weeks, individual mice had as a humane endpoint. As shown in Figure 2(b), no to be euthanized due to reaching the humane endpoint mice in the STZ-injected group given softened chow of 20% weight-loss, making statistical comparisons of reached this humane endpoint. In contrast, 37.5% of mean body weight change for the whole study biased by the mice in the pellet group were euthanized due to the loss of mice with the most significant weight loss. 20% weight loss and this was despite the increased fre- The body weight change for the entire study can be quency of supportive measures. Most of these mice found in Supplementary Figure 3. were euthanized between day 50 and termination.

(a) (b) 80 4 ns ns * *

3 60

2 40

1 20 Feces weight (g) conc. (ng/g feces)

0 Corticosterone metabolites 0 Vehicle, Vehicle, STZ, STZ, Vehicle, Vehicle, STZ, STZ, pellets softened food pellets softened food pellets softened food pellets softened food

150

100

50 metabolites (ng/19h)

Total faecal corticosterone 0 Vehicle, Vehicle, STZ, STZ, pellets softened food pellets softened food

Figure 3. Water-softened chow reduces faecal corticosterone metabolite excretion. (a) Total weight of faeces samples taken from the metabolic cages at week 6 after STZ injections. Data are shown as individual points as well as mean SD, *p ¼ 0.0156, by one-way ANOVA. (b) Corticosterone metabolite concentration in faeces in ng/g faeces. Data are shown as individual points as well as mean SD, *p ¼ 0.0449, by one-way ANOVA. (c) Total corticosterone metabolites excreted in the faeces during 19 h in metabolic cages. Data are shown as individual points as well as mean SD, ***p ¼ 0.0007, by one-way ANOVA. 378 Laboratory Animals 52(4)

It should be mentioned that three diabetic mice receiv- the length of the upper and lower incisors was mea- ing softened chow were found dead (on day 30, 95 and sured at termination (Supplementary Figure 4). No sig- 97) without showing any increased weight loss or sign nificant difference was found in the length of either the of discomfort in the preceding days. No unambiguous upper or the lower incisors, suggesting that gnawing reasons were found for these spontaneous deaths behaviour and teeth-wear was intact despite the supple- during necropsy. One mouse had a slightly increased ment of water-softened chow. spleen and another one signs of mild ascites. In the last case, the decay process made in impossible to Corticosterone excretion reach a diagnosis. We find it unlikely that the softened food caused these spontaneous deaths, although we To assess the general level of stress experienced by the cannot completely rule this out. mice, faecal corticosterone metabolites were measured As a behavioural measure and to investigate if the in the mice. Faecal samples were taken at week 6 from mice receiving softened chow were still normally wear- the metabolic cages as a collection of the total excretion ing their teeth eating pellets or gnawing wooden blocks, over 19 h and this was analysed for corticosterone

(a) (b) 10 40 STZ

8 ns 30 ** 6 20 Diabetes criteria 4

% HbA1c 16,6 mmol/L

10 2 Blood glucose (mmol/L)

0 0 0 50 100 0 5 10 15 20 25 Time (days) Time (day)

40 STZ STZ, pellets STZ, softened food

30 ***

20 Diabetes criteria 16,6 mmol/L

10 Blood glucose (mmol/L)

0 0 20406080100 Time (day)

Figure 4. Diabetes induction. (a) Percent HbA1c measured in tail vein blood every third week. Data are shown as mean SD. p ¼ 0.2356, by comparing AUC in a two-tailed students t-test. (b) BG measured from tail blood twice weekly. Data shown from day 1 (prior to STZ injection) until the first occurrence of a euthanization at day 22. Mean SD. **p ¼ 0.0095, by comparing AUC in a two-tailed students t-test. (c) BG for entire study. Mice euthanized during the study are excluded completely from these data. Mean SD. ***p ¼ 0.0003, by comparing AUC in a two-tailed students t-test. Nørgaard et al. 379

(a) (b) *** 2000 ** 3 *** **** ns ns 1500 ns 2 ns lbumin 1000

Urinary A 500 excretion rate (ug/24h) Relative kidney weight (% of total body weight) 0 0 Vehicle, Vehicle, STZ, STZ, Vehicle, Vehicle, STZ, STZ, pellets softened food pellets softened food pellets softened food pellets softened food

Figure 5. Disease readouts. (a) 24 h urinary albumin excretion rate calculated from urine collected in metabolic cages 14 weeks after STZ injection. Data are shown as individual points as well as mean SD. **p ¼ 0.0013, ***p ¼ 0.0002, by one- way ANOVA. (b) Kidney weight measured at termination and presented as relative kidney weight as a percentage of body weight. Data are shown as individual points as well as mean SD. ***p ¼ 0.0006, ****p < 0.0001, by one-way ANOVA. content. The total weight of the faeces samples showed determine the gross pathological impact on kidneys that the diabetic mice only receiving pellets defecated following diabetes induction, the kidney-to-body significantly more in the 19 h compared to the diabetic weight ratio was measured at termination in all mice given softened chow (Figure 3(a)). The concentra- groups (Figure 5(b)). Compared to vehicle groups, tion of corticosterone per g faeces was calculated both STZ-treated groups showed a significant increase and showed that the diabetic mice receiving softened in relative kidney weight. The kidney weight at termin- chow had a significantly lower faecal corticosterone ation could therefore further indicate if the softened concentration compared to the group only receiving chow affects the induction of DN. The softened chow pellets (Figure 3(b)). When looking at the total did not affect the kidney weight in either the vehicle- or amount excreted during the 19 h, the diabetic mice STZ-treated mice. receiving softened chow excreted significantly lower total amount of corticosterone metabolites in their Discussion and conclusion faeces compared to the diabetic mice only eating pellets (Figure 3(c)). The STZ-induced diabetic mouse model is one of the most used models for studying diabetes and DN. Diabetic and DN disease readouts However, challenges regarding significant weight loss and euthanization rates still calls for optimization and To investigate if the supplementation of water- refinement of this model to improve overall animal wel- softened chow might affect the induction of diabetes, fare and study quality. In this study, we found that BG was measured twice weekly, and%HbA1c giving the mice the opportunity to eat water-softened every third week (Figure 4). As expected both BG chow in addition to the regular chow pellets could and %HbA1c was increased in both STZ-treated reduce weight loss in STZ-injected male mice. We groups compared to the vehicle groups. When compar- found a significant reduction in both the acute weight ing the two STZ-treated groups, a significant higher BG loss (first 3 weeks) and in the number of mice that is found in the group receiving softened food both in reached the humane endpoint of 20% weight loss. the first 3 weeks and the entire study (Figure 4(a) and The latter was found even though the STZ-injected (b)). This difference is not seen when comparing the mice receiving softened chow received a significantly HbA1c for these groups, and both results suggest an lower frequency of supportive treatments with insulin intact induction of diabetes (Figure 4(c)). and saline throughout the study, without which, the The 24 h urinary albumin excretion rate (UAER) difference might have been even more significant. was calculated from urine samples after 19 h in meta- Overall, this study demonstrates that supplementing bolic cages 14 weeks after STZ (Figure 5(a)). This STZ-injected male mice with water-softened chow readout showed no significant difference when the once daily in addition to regular chow pellets could water-softened chow supplement was compared to significantly improve animal well-being. Since only chow pellets alone in the STZ groups, suggesting an male mice were included in this study, it is therefore intact induction of albuminuria. Furthermore, to only possible to conclude on the effect of softened 380 Laboratory Animals 52(4) food on diabetic male mice. In this study, we looked excretion (Figure 3(c)), this should be interpreted with primarily on weight loss but in future studies it would caution. It seems, though, that the softened food sup- be interesting to make a thorough and systematic inves- plement to STZ-treated animals under no circum- tigation of the nature of the weight loss, behaviour, stances are resulting in higher corticosterone levels, appearance, body condition score etc. and does thus not seem to inflict any negative impact It is difficult to get a comprehensive picture of the to the welfare of the animals. Furthermore, the mice extent of the issues with weight loss and loss of animals were housed in the metabolic cages for 19 h to obtain in this model, since many publications do not account urine samples for albuminuria measurement and faeces for the number of animals lost during these studies.14 samples for measurements of corticosterone metabol- As described in other publications, we in this study ites. Since the gastrointestinal transit time is only used injections with insulin and saline as supportive around 6 h in mice,36 the higher total excretion of cor- treatment. We have found this to minimize the loss of ticosterone in the STZ-injected mice only eating pellets animals especially during longer-term studies such as could both be an effect of an increased stress level in for development of measurable nephropathy. Other both the home cage and in the metabolic cages. It has researchers have suggested that liquid nutrition supple- previously been shown that being housed in metabolic ments could be used in combination with insulin in cages significantly increases the excretion of cortico- order to prevent significant weight loss in this model,9 sterone metabolites,37 although that study investigated but how this supplement affect the diabetic mice is mice constantly housed in metabolic cages for several unknown and would be an interesting topic for further weeks. Since the mice in this study were only housed in studies. In the current study, we present an initiative the metabolic cage for 19 h, it is not possible to con- that could potentially reduce weight loss and euthani- clude on the effect of the metabolic cages on the level of zation rate by simply giving the mice a water-softened stress in the different groups. The increased level of chow supplement in addition to their regular chow. stress hormone in the STZ-injected mice only eating Importantly, giving the mice water-softened chow pellets could also be the result of increased stress once daily did not affect the induction or magnitude prior to placement in the metabolic cage. of diabetes or change their DN phenotype, suggesting The addition of the water-softened chow does not an intact disease model. Our results indicate that giving interfere with the disease induction or progression. the mice the opportunity of eating softened chow helps We detect slightly higher BG levels throughout the stabilize their weight. And by reducing weight loss, the study in the STZ injected group receiving water- need for supportive treatment is also lowered. This sig- softened chow, but this increase is not large enough nificantly lowers the risk of stressing the animals due to to be reflected in the HbA1c measurements. Also the frequent weighing and injections and the work load of UAER increase was comparable in the two STZ groups monitoring and handling them. displaying an intact induction of both diabetes and Measuring faecal levels of corticosterone metabolites albuminuria. is widely used as a biomarker of stress and has some From our experience the STZ-induced diabetes advantages compared to quantifying blood levels of model is a model prone to variability; also when corticosterone, since it gives a measure of stress levels it comes to weight loss. Therefore, even though the over a longer period and is not affected by the potential results of this study show a clear effect of giving the stress response of the blood sample procedure.30–33 STZ-injected mice softened chow it might be appropri- While the concentration of corticosterone metabolites ate to test the reproducibility of these results in other in the STZ-injected mice receiving softened chow sup- research environments. Furthermore, it would be rele- plement was only slightly lower than in those getting vant to explore the effect of the softened food on other only chow pellets, this difference was even greater when strains used in DN research as well as female mice and looking at the total corticosterone metabolites excreted additionally when using a different STZ injection during the 19 h, suggesting a lower stress response protocol. Currently, this initiative has only been in the water-softened chow group. It has previously assessed in the STZ-induced diabetes/DN model but been argued that expressing the corticosterone metab- could potentially be considered in other mouse olites as total amount excreted during a time period is models where high weight loss is an issue. more accurate than concentration.29,34,35 Therefore, After first being described in 1959 by Russell and one could interpret the data as if the STZ-injected Burch, the concept of the 3Rs now has become the mice receiving softened food were less stressed than most important ethical principle when working with the STZ- injected mice receiving pellets. However, animal welfare in research. In this study we have been since the vehicle groups differ in proportion to the implementing both the refinement and reduction part of STZ groups in terms of corticosterone levels these principles.38 By reducing weight loss and lowering expressed as concentration (Figure 3(b)) versus the need for supportive insulin and saline this initiative Nørgaard et al. 381 is significantly refining this animal model. Furthermore, 7. Lenzen S. The mechanisms of alloxan- and streptozoto- by giving the mice softened chow we reduce number cin-induced diabetes. Diabetologia 2008; 51: 216–226. of animals needed in studies as fewer animals reach 8. Szkudelski T. The mechanism of alloxan and streptozo- significant weight loss and the humane endpoint. tocin action in B cells of the rat pancreas. Physiol Res While the supplement of water-softened chow increased 2001; 50: 537–546. 9. Tesch GH and Allen TJ. Rodent models of streptozoto- the overall animal welfare; it did not affect the mea- cin-induced diabetic nephropathy. Nephrology 2007; 12: sured readouts for diabetes or DN which indicates an 261–266. unchanged disease model. 10. Leiter EH. Multiple low-dose streptozotocin-induced In conclusion, this study suggests that giving hyperglycemia and insulitis in C57BL mice: influence of STZ-injected male mice water-softened chow once inbred background, sex, and thymus. Proc Natl Acad Sci daily in addition to normal chow pellets could signifi- USA 1982; 79: 630–634. cantly reduce weight loss and reduce the risk of losing 11. Ostenson CG, Grill V and Roos M. Studies on sex animals to humane endpoints without interfering with dependency of B-cell susceptibility to streptozotocin in the development of diabetes or DN. a rat model of type II diabetes mellitus. Exp Clin Endocrinol 1989; 93: 241–247. 12. Gurley SB, Clare SE, Snow KP, et al. Impact of genetic Acknowledgements background on nephropathy in diabetic mice. Am J The authors would like to thank the laboratory technicians in Physiol Renal Physiol 2006; 290: F214–F222. Diabetes Complications Pharmacology for assistance in the 13. Le May C, Chu K, Hu M, et al. Estrogens protect pancre- in vivo part of the experiment as well as the Laboratory atic beta-cells from apoptosis and prevent insulin- Animal Science group at Novo Nordisk A/S for assistance deficient diabetes mellitus in mice. Proc Natl Acad Sci with mouse husbandry. Also, the authors thank Helle USA 2006; 103: 9232–9237. Runchel Porsdal and Trine Marie Ahlman Glahder for assist- 14. Deeds MC, Anderson JM, Armstrong AS, et al. Single ance with measurements of corticosterone metabolites. dose streptozotocin-induced diabetes: considerations for study design in islet transplantation models. Lab Anim Declaration of Conflicting Interests 2011; 45: 131–140. 15. Yorek MA. Alternatives to the streptozotocin-diabetic The author(s) declared no potential conflicts of interest with rodent. Int Rev Neurobiol 2016; 127: 89–112. respect to the research, authorship, and/or publication of this 16. Dekel Y, Glucksam Y, Elron-Gross I, et al. Insights into article. modeling streptozotocin-induced diabetes in ICR mice. Lab Anim 2009; 38: 55–60. Funding 17. Breyer MD, Bottinger E, Brosius FC 3rd, et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2005; The author(s) disclosed receipt of the following financial sup- 16: 27–45. port for the research, authorship, and/or publication of this 18. Weiss RB. Streptozocin: a review of its pharmacology, news article: This work was a part of a PhD project funded by efficacy, and toxicity. Cancer Treat Rep 1982; 66: Innovation Fund Denmark (grant number 4135-00008B) and Novo Nordisk A/S. 427–438. 19. Tay YC, Wang Y, Kairaitis L, et al. Can murine diabetic nephropathy be separated from superimposed acute renal References failure? Kidney Int 2005; 68: 391–398. 1. Kitada M, Ogura Y and Koya D. Rodent models of dia- 20. Palm F, Ortsater H, Hansell P, et al. Differentiating betic nephropathy: their utility and limitations. Int J between effects of streptozotocin per se and subsequent Nephrol Renovasc Dis 2016; 9: 279–290. hyperglycemia on renal function and metabolism in the 2. Like AA and Rossini AA. Streptozotocin-induced pancre- streptozotocin-diabetic rat model. Diabetes Metab Res atic insulitis: new model of diabetes mellitus. Science 1976; Rev 2004; 20: 452–459. 193: 415–7. 21. Kume E, Fujimura H, Matsuki N, et al. Hepatic 3. Vavra JJ, Deboer C, Dietz A, et al. Streptozotocin, a new changes in the acute phase of streptozotocin (SZ)- antibacterial antibiotic. Antibiot Annu 1959; 7: 230–235. induced diabetes in mice. Exp Toxicol Pathol 2004; 55: 4. Schein PS, O’Connell MJ, Blom J, et al. Clinical antitumor 467–480. activity and toxicity of streptozotocin (NSC-85998). 22. Piyachaturawat P, Poprasit J, Glinsukon T, et al. Gastric- Cancer 1974; 34: 993–1000. mucosal lesions in streptozotocin-diabetic rats. Cell Biol 5. Rakieten N, Rakieten ML and Nadkarni MR. Studies on Int Rep 1988; 12: 53–63. the diabetogenic action of streptozotocin (NSC-37917). 23. Hayashi K, Kojima R and Ito M. Strain differences in the Cancer Chemother Rep 1963; 29: 91–98. diabetogenic activity of streptozotocin in mice. Biol 6. Cohen MP and Klein CV. Glomerulopathy in rats with Pharm Bull 2006; 29: 1110–1119. streptozotocin diabetes. Accumulation of glomerular base- 24. Brosius FC III, Alpers CE, Bottinger EP, et al. Mouse ment membrane analogous to human diabetic nephropa- models of diabetic nephropathy. J Am Soc Nephrol 2009; thy. J Exp Med 1979; 149: 623–631. 20: 2503–12. 382 Laboratory Animals 52(4)

25. Brosius FC, III. Diabetic Complications Consortium. noninvasive technique to monitor stress hormones. Low-Dose Streptozotocin Induction Protocol (Mouse). Hormones Behav 2004; 45: 10–22. https://www.diacomp.org/shared/showFile.aspx?doc- 32. Mostl E and Palme R. Hormones as indicators of stress. typeid¼3&docid¼19 (accessed 20 December 2017). Domest Anim Endocrinol 2002; 23: 67–74. 26. O’Brien BA, Harmon BV, Cameron DP, et al. Beta-cell 33. Kalliokoski O, Hau J, Jacobsen KR, et al. Distribution apoptosis is responsible for the development of IDDM in and time course of corticosterone excretion in faeces and the multiple low-dose streptozotocin model. J Pathol urine of female mice with varying systemic concentra- 1996; 178: 176–181. tions. Gen Comp Endocrinol 2010; 168: 450–454. 27. Eizirik DL and Mandrup-Poulsen T. A choice of death: 34. Kalliokoski O, Teilmann AC, Abelson KS, et al. The the signal-transduction of immune-mediated beta-cell distorting effect of varying diets on fecal glucocorticoid apoptosis. Diabetologia 2001; 44: 2115–2133. measurements as indicators of stress: a cautionary dem- 28. Meier M, Park JK, Overheu D, et al. Deletion of protein onstration using laboratory mice. Gen Comp Endocrinol kinase C-beta isoform in vivo reduces renal hypertrophy 2015; 211: 147–153. but not albuminuria in the streptozotocin-induced dia- 35. Hau J, Kalliokoski O, Jacobsen K, et al. Interpretations betic mouse model. Diabetes 2007; 56: 346–354. of faecal concentrations of corticosteroids. Lab Anim 29. Sundbom R, Jacobsen KR, Kalliokoski O, et al. 2011; 45: 129–130. Post-operative corticosterone levels in plasma and feces 36. Padmanabhan P, Grosse J, Asad A, et al. of mice subjected to permanent catheterization and auto- Gastrointestinal transit measurements in mice with mated blood sampling. In Vivo 2011; 25: 335–342. Tc-99m-DTPA-labeled activated charcoal using 30. Palme R, Rettenbacher S, Touma C, et al. Stress hor- NanoSPECT-CT. EJNMMI Res 2013; 3: 8. mones in mammals and birds: comparative aspects 37. Kalliokoski O, Jacobsen KR, Darusman HS, et al. Mice regarding metabolism, excretion, and noninvasive meas- do not habituate to metabolism cage housing-a three urement in fecal samples. Ann NY Acad Sci 2005; 1040: week study of male BALB/c mice. PLoS One 2013; 8: 162–171. e58460. 31. Touma C, Palme R and Sachser N. Analyzing cortico- 38. Russell WMS and Burch RL. The principles of humane sterone metabolites in fecal samples of mice: a experimental technique. London: Methuen, 1959.

Re´sume´ La souris diabe´tique induite par streptozotocine (STZ) est un mode`le de diabe`te et de ne´phropathie diabe´tique (ND) largement utilise´. Il est cependant bien connu que ce mode`le est sujet a` une importante perte de poids, qui en de´pit de mesures de soutien, engendre souvent des taux d’euthanasie. Pour surmonter ces proble`mes, nous nous sommes demande´ si donner une nourriture ramollie par de l’eau en plus de leur nourriture normale aux souris diabe´tiques induites par STZ diminuerait la perte de poids, la nećessite´ d’un traitement adapte´ et le nombre de souris atteignant le taux e´thiquement acceptable de 20% de perte de poids. Lors d’une e´tude de 15 semaines meneé sur la ND induite par STZ, nous avons de´montre´ que les souris maˆles diabe´- tiques recevant une nourriture ramollie pre´sentaient une re´duction de la perte ponde´rale aigue¨ apre`s traitement STZ (p ¼ 0,045). En outre, moins de souris ont e´te´ euthanasieés en raison de la perte de poids. En comple´tant la nourriture normale des souris diabe´tiques par de la nourriture ramollie, aucune souris n’a atteint 20% de perte de poids alors que 37,5% des souris sans ce supple´ment ont atteint ce niveau e´thiquement acceptable (p ¼ 0,0027). L’excre´tion des me´tabolites de la corticoste´rone dans les fe`ces a e´te´ re´duite chez des souris diabe´tiques recevant de la nourriture ramollie (p ¼ 0,0007), ce qui sugge`re un niveau de stress geńe´ral moins e´leve´. Enfin, il a e´te´ de´montre´ qu’un supple´ment de nourriture ramollie par de l’eau n’affectait pas significativement l’induction de la maladie c.-a`-d. les parame`tres cle´s de %d’HbA1c et d’albuminurie et n’entraıˆnait pas non plus une usure anormale des dents. En conclusion, l’ajout d’une nourriture ramollie raffine le mode`le de souris diabe´tiques induites par STZ en re´duisant conside´rablement le stress, la perte de poids et le nombre d’animaux sacrifie´s en raison de limites e´thiquement acceptables, tout en maintenant les pheńotypes cle´s du diabe`te et de la ne´phropathie.

Abstract Die Streptozotocin (STZ)-induzierte diabetische Maus ist ein weit verbreitetes Modell fu¨r Diabetes und diabetische Nephropathie (DN). Ein mit diesem Modell bekanntermaßen einhergehendes Problem ist jedoch der erhebliche Gewichtsverlust, der trotz unterstu¨tzender Maßnahmen ha¨ufig zu hohen Einschla¨ferungsquoten fu¨hrt. Um diese Probleme zu bewa¨ltigen, legten wir die Annahme zugrunde, dass die Verabreichung von in Wasser erweichtem Futter zusa¨tzlich zu normalem Futter an STZ-induzierte diabetische Ma¨use den Nørgaard et al. 383

Gewichtsverlust reduzieren, die Notwendigkeit einer unterstu¨tzenden Behandlung senken und die erforder- liche Anzahl von Maüsen zur Erreichung des humanen Endpunkts von 20% Gewichtsverlust reduzieren wu¨rde. In einer 15-wo¨chigen STZ-induzierten DN-Studie wiesen wir nach, dass der akute Gewichtsverlust bei diabetischen ma¨nnlichen Maüsen, die weiches Futter erhielten, nach einer STZ-Behandlung reduziert wurde (p ¼ 0,045) und außerdem weniger Maüse aufgrund von Gewichtsverlust eingeschla¨fert wurden. Die Gabe von weicherem Futterzusatz an diabetische Maüse resultierte bei keinem Tier in einem Gewichtsverlust von 20%, wa¨hrend 37,5% der Maüse ohne diese zusa¨tzliche Verabreichung diesen humanen Endpunkt erreichten (p ¼ 0,0027). Die Ausscheidung von Corticosteron-Metaboliten im Stuhl wurde bei mit weichem Futterzusatz behandelten diabetischen Maüsen reduziert (p ¼ 0,0007), was auf weniger allgemeinen Stress hindeutet. Schließlich konnte gezeigt werden, dass der mit Wasser entha¨rtete Futterzusatz sich nicht signifikant auf die Induzierung wichtiger Krankheitsparameter, d. h. % HbA1C und Albuminurie, auswirkte, und auch keinen abnormalen Zahnverschleiß zur Folge hatte. Zusammenfassend la¨sst sich sagen, dass die zusa¨tzliche Gabe von weicher Nahrung das STZ-induzierte diabetische Mausmodell signifikant verbessert, indem Stress, Gewichtsverlust und die Anzahl der Tiere, die fu¨r humane Endpunkte geopfert werden, gesenkt werden, wa¨hrend die wesentlichen Pha¨notypen diabetischer Nephropathie erhalten bleiben.

Resumen El ratoń diabe´tico tratado con estreptozotocina es un modelo de diabetes y nefropatıá diabe´tica utilizado habitualmente. No obstante, se sabe que este modelo padece una gran pe´rdida de peso, que a pesar de tomar medidas de soporte, a menudo provoca un alto grado de animales sacrificados. Para hacer frente a estos problemas, supusimos que suplementando la comida de los ratones diabe´ticos tratados con estreptozotocina con comida ablandada con agua reducirıálape´rdida de peso y la necesidad de un tratamiento adicional, adema´s de reducir el nu´mero de ratones que alcanzan el lı´mite humano de pe´rdida de peso del 20%. En un estudio de 15 semanas de nefropatıá diabe´tica tratada con estreptozotocina, demostramos que los ratones macho diabe´ticos que recibıán comida ablandada habıá reducido la pe´rdida de peso grave tras el tratamiento con estreptozotocina (p ¼ 0,045) y, por tanto, se sacrificaron menos ejemplares de ratones por pe´rdida de peso. Al suministrar a los ratones diabe´ticos comida ablandada, ninguń ratoń llego´ a una pe´rdida de peso del 20% mientras que el 37,5% de los ratones sin este tipo de comida blanda llegaron al lı´mite humano (p ¼ 0,0027). La excrecioń de metabolitos de corticosterona en las heces se redujo en los ratones diabe´ticos que comıán alimentos blandos (p ¼ 0,0007), lo que sugerıá unos niveles inferiores de estre´s general. Finalmente, se demostro´ que el suplemento de comida ablandada con agua no afecto´ significativamente la induccioń de los para´metros clave de enfermedad, es decir, %HbA1C y albuminuria, ni tampoco provoco´ un desgaste dental anormal. En conclusioń, el suplemento de comida ablandada esta´ refinando el modelo de ratoń diabe´tico tratado con estreptozotocina significativamente reduciendo el estre´s, la pe´rdida de peso y el nu´mero de animales sacrificados debido a lı´mites humanos, pero manteniendo a su vez los fenotipos clave de diabetes y nefropatıá. laboratory an imals Original Article limited Laboratory Animals 2018, Vol. 52(4) 384–393 ! The Author(s) 2018 Partial cage division significantly Reprints and permissions: sagepub.co.uk/ reduces aggressive behavior in journalsPermissions.nav DOI: 10.1177/0023677217753464 male laboratory mice journals.sagepub.com/home/lan

Bret R Tallent1,3, L Matthew Law1,2,3, Rachel K Rowe1,2,3 and Jonathan Lifshitz1,2,3

Abstract Aggression in mice often results in injury leading to unplanned euthanasia or the initiation of protocols to isolate animals, thereby increasing research costs and straining resources. Here, we tested if adding a partial cage divider into existing mouse cages affected aggressive-like behavior in group-housed male mice (18 mice; 3 per cage). Mice were randomly assigned to one of two groups upon arrival to the vivarium: (1) standard cage; (2) cage with a partial cage divider. Behavioral observation over 12 hours were conducted at day one, two, and seven after receipt at the facility in order to assess aggression during the course of establishing dominance hierarchies. Observers blinded to study design and hypothesis scored each video for the number and type of aggressive behaviors, which were summed for each hour and analyzed. Results indicated a statistically significant decrease in aggressive behaviors of mice in cages with dividers compared to mice in standard cages. We conclude that cage dividers, which resemble burrows and provide access to common food/water, may promote rigorous research by reducing the number of animals used in a study and refining housing, thus, improving animal welfare.

Keywords aggression, mouse, cage divider, enrichment, housing, fighting

Date received: 31 July 2017; accepted: 20 December 2017

Introduction limited. Aggressive interactions typically begin with a Mice have become essential to biomedical and bio- tail rattle. If this warning is not heeded, it is usually logical research since they were first taken out of the followed by a frontal attack wherein the dominant wild and domesticated in the laboratory. In the wild, male chases the ‘‘intruder’’ until line of sight is mice typically live in burrows during daylight hours broken or the intruder leaves the territory.8–10 and spend nighttime hours exploring their environ- In the laboratory, federal guidelines regulate cage ment. Mice are a social species and choose to cohabit size and density, where typical home cages are with other mice, thereby forming demes (territory maintained by a family group).1,2 Deme sizes depend 1 upon available resources in the territory, and influence Department of Child Health, University of Arizona College of Medicine-Phoenix, Phoenix, AZ, USA the number of females and subordinate males asso- 2Phoenix VA Healthcare System, Phoenix, AZ, USA ciated with a dominant male. The dominant male will 3Barrow Neurological Institute at Phoenix Children’s Hospital, chase unfamiliar males from the deme, while tolerating Phoenix, AZ, USA juvenile and subordinate males. The highest tolerance levels are found in densely populated areas with an Corresponding author: 3–7 Bret R Tallent, Translational Neurotrauma Research Program, abundance of food. Under laboratory conditions of Department of Child Health, University of Arizona College of controlled resources, investigators set the deme size and Medicine-Phoenix, Phoenix, AZ 85004, USA. tolerance for unfamiliar males by the dominant male is Email: [email protected] Tallent et al. 385 transparent, barren habitats, appropriately called breeding animals.26,37–39 These are different from the ‘‘shoebox cages.’’ These commercial cages meet the rec- visible burrow system (VBS) that has been used to ommendations of the ‘‘Guide for the Care and Use of study stress in mice,40 in that they are a ‘‘home cage’’ Laboratory Animals’’ as to floor space and population feature and not a test apparatus, and they allow for densities, based upon minimal floor area per weight of multiple ‘‘burrows.’’ Mice reared in these systems each mouse.11 Typical housing consists of either a gained more body weight, had lower adrenal weights, standard cage or a breeder cage. Standard cages aver- and were more active than animals reared in an open age 500 cm2 of floor space, and house demes of up to cage.26 In an open field behavior observation, mice five mice per cage. Breeder cages average 1200 cm2 of from a complex cage system had increased ambulation floor space, and house a female dam with her litter, up and decreased defecation, indicating lower emotional- to ten mice per cage. ity, or reactivity.12 Emotionality and reactivity are syn- This barren, confined space provides minimal, if any, onymous, referring to a complex phenotype observed in stimulation or opportunity for natural behaviors such prey animals in response to a variety of situations. as exploration, burrowing, or hiding. Additionally, The variance in reactivity between mice (cage mates social dominance is an inherent behavior of mice and or other) may explain the large group sizes needed to under laboratory conditions a dysfunctional dominance achieve statistical significance. hierarchy, in which social cues and escape or submissive In the present study, we custom-designed cage div- behaviors cannot be expressed, may be estab- ider inserts that create complex cage systems without lished.4,5,7,12–14 As a result, the mouse behavior likely permanent modification to existing caging units. These drifts from natural expression of innate behavior inserts divided half of the cage into thirds, with each towards the abnormal as the deme welfare becomes emulating above ground burrows or nests and allowed compromised.15,16 Mice housed in shoebox cages may for open access to a common area for food and water. be unable to respond in socially appropriate ways to The square inches of each burrow are approximately each other, and subordinate mice are unable to flee equal, where the burrow walls were either divider from the dominant mouse.10,16 This inhibition of material or the home cage. The cage dividers added innate behavior can cause a state of suffering in the to cage complexity and increased wall space, which subordinate mouse, and an increase in aggression appeals to a rodent’s inherent thigmotactic (wall-seek- from the dominate mouse.16 Increased aggressive ing) behavior.43 The objective of the study was to test if behavior affects the psychological and physiological the addition of a partial cage divider upon arrival to the welfare of the subordinate mice, either by aggravating vivarium decreased aggressive behavior in mice. We an appropriate behavioral response or through pain hypothesized that the addition of a partial cage divider and distress resulting from injuries.17 would significantly decrease aggressive behaviors com- The typical treatments for a cage of aggressive ani- pared to mice housed in a standard, non-divided cage. mals are the addition of environmental enrichment Overall, our results indicated a significant decrease in devices to each cage,18–21 isolation of animals, or aggressive behavior and fighting in mice. These data euthanasia. Isolation of animals can further alter support the addition of cage dividers to improve overall physiological parameters, creating issues with variabil- animal welfare. ity in experimental results and scientific validity,10,22–25 not to mention additional costs to the investigator. Animals Devices such as nesting materials and nest boxes are intended to provide complexity to the cage, allow- Male Balb/c mice (8 weeks old) (Envigo, Inc., ing mice to express some natural behaviors and Indianapolis, IN) were used for all experiments increase the psychological perception of space.19,26 (n ¼ 18). This line was selected because they are Unfortunately, while some enrichments have shown commonly used, somewhat aggressive, and white in a positive effect on animals,22–31 others have color (for easier tracking of individuals). Mice were demonstrated an increase in aggressive behavior and housed in a 10 h light/14 h red light cycle at a constant indeterminate effects on both behavior and temperature (23 2C) with food and water physiology.10,16,20,32–42 available ad libitum. Routine husbandry included Few published studies focus on cage dividers or com- daily evaluation and documentation of each animal’s plex caging for mice.26,37–39 Mice reared in a complex condition. Cage changes occurred every other cage system that emulates a burrow-like environment week (none during the course of evaluation). At the are healthier and less reactive compared to those in conclusion of this study, mice were transferred to standard housing.26 Complex cage systems include another active IACUC (Institutional Animal Care large cages, or caging systems with permanent modifi- and Use Committee) protocol for inclusion in cations to establish 5–9 burrow-like dividers for additional studies. 386 Laboratory Animals 52(4)

Materials and methods had visible wounds requiring veterinarian intervention. Study design No animals or cages were excluded from the study. Data sets were screened using the extreme studentized In a controlled laboratory experiment, this study tested deviate method for significant outliers. No significant the benefits of adding novel cage dividers into standard outliers were excluded from the analyses. mouse caging without modification on aggressive behavior between group-housed mice (Figure 1). For Cage dividers this study, we used three animals per cage as the divider created a three-burrow partition. We selected three Cage dividers were hand-fabricated from opaque, cages for each group (standard caging; divided white, b-flute corrugated plastic. They were designed caging), establishing a total n ¼ 6 for statistical com- to fit inside existing cages (InnocageÕ Mouse Cage, parisons (see below). All animal studies were conducted Innovive, San Diego, CA; outside dimensions 14.7’’ at AAALAC International accredited facilities in L 9.2’’ W) without modification to the enclosure or accordance with the guidelines established by the inter- obstructing ventilation. Dividers were held in place by nal IACUC at the University of Arizona (UA #15-075) the feed hopper and cage lid, effectively dividing half of and the NIH guidelines for the care and use of labora- the cage into thirds (Figure 1), from cage floor to cage tory animals. Upon arrival to the animal facility from lid. All four compartments (common area and three the commercial vendor, 18 animals were randomly burrows) were open to one another and accessible. assigned to cages until three animals were in each par- The divider length was 7’’ and each burrow was tially divided or undivided cage. These are the first data approximately 3’’ wide. Total floor space (522.58 cm2) to evaluate cage dividers with respect to aggressive was unaffected by the addition of the cage divider. The behavior, and so only a post-hoc power analysis walls of the cage divider ran parallel to the long axis of could be conducted to confirm that the group sizes the cage, maintaining line-of-sight for daily observation were sufficient for statistically robust detection of dif- and health checks by animal care personnel. ferences in aggressive behavior between groups (divided and standard). Data collection stopped at pre-deter- Housing mined final endpoints based on days in cage with or without dividers. Pre-determined exclusion criteria Upon receipt into the vivarium from the vendor, mice included removing any animal from the study that were randomly divided into two groups: group 1 was

Figure 1. Standard Innovive cage with divider, as seen from rear (a), side (b), and front (c). A schematic floorplan of a rectangular mouse cage with the cage divider showing half of the cage divided into thirds (burrows 1–3) and the remaining half cage as a great room with access to food and water. See text for dimensions. Tallent et al. 387 Statistical analysis housed in standard caging without dividers (Figure 2(a)); group 2 was housed in standard caging Data are shown as mean SEM and analyzed using with the addition of cage dividers (Figure 2(b)). SPSS, with statistical significance assigned when All cages in both groups had a single water bottle, p < 0.05, unless otherwise indicated. Number of events feed hopper, corn cob bedding, and a nestlet square, were analyzed using a repeated measure two-way ana- in accordance with standard procedures for the animal lysis of variance (ANOVA; group time or type) fol- facility. A total of 3 cages per group, containing 3 mice lowed by post-hoc tests, applying a Bonferroni per cage were included in the study. correction to account for multiple comparisons. For all data, the assumption that data were normally distributed and held homogeneous variance were verified (Levene’s Video recordings and aggressive behavior test for homogeneity or Mauchly’s test of sphericity) to scoring ensure the validity of the statistical approaches used. Statistical values are included in the results. A video camera (Sony Handycam HDR-CX190 HD low light) was positioned at the rear of each cage to Results continuously record behavior. Recordings (color, 1920 1080/60i quality, 24 Mbps) were made in 12 h All cages and mice were included in the study of aggres- sessions (maximum capacity of digital storage) starting sive behavior between divided and standard cages. No upon receipt in the vivarium (day 1; acclimation unforeseen outcomes occurred. We recorded mouse period), 24 h later (day 2; establishment of the hier- behavior for 6 h during the light cycle followed by 6 h archy), and one week after receipt (day 7; stable hier- during the dark cycle on days 1, 2, and 7 after initial archy). All recordings began with 6 h remaining in the arrival to the vivarium. We tallied the number of light cycle and continued 6 h into the dark cycle. aggressive behavior events within each group (standard Recordings occurred between 12:00 and 24:00 on each cage, divided cage), by type (posturing to severe), over day which is after the daily health check and any cage time. Data for time of day and aggression type were changes that occur in the housing room. The two initial analyzed independently. recordings provided data during acclimation to the new environment, while the third recording captures the Cage dividers decreased aggressive established hierarchy and status quo for the cage. behavior in light and dark cycles Offline, recordings were observed for aggressive behavior. Two independent observers tallied the Mice housed in divided cages had significantly fewer number and type of aggressive behaviors for each aggressive behavior events than mice housed in stand- hour in each cage (total 216 h). Aggressive behavior ard cages, in terms of daily combined number of events type was recorded on a 1–5 scale, where: 1 ¼ posturing; (F(1,4) ¼ 280.189, p < 0.001), events in the light cycle 2 ¼ scuffle/fight; 3 ¼ unprovoked biting; 4 ¼ blood (F(1,4) ¼ 38.822, p < 0.01), and events in the dark (in cage or on animal); 5 ¼ severe injury (wherein cycle (F(1,4) ¼ 289.053, p < 0.001) (Figure 3). There was animal needs immediate attention). The number of an overall effect of day for aggressive behavior with sig- aggressive behavior events were summed within each nificantly more daily combined events (F(2,8) ¼ 19.840, hour, over the three observation days, and collated p < 0.01), light cycle events (F(2,8) ¼ 8.915, p < 0.01), and within each group (standard cage, divided cage) dark cycle events (F(2,8) ¼ 31.255, p < 0.001), on day 2 and analyzed. compared to day 1 and day 7.

Figure 2. Cage of three Balb/c mice with cage divider (a) and without cage divider (b). Note the minimal obtrusion of the cage divider in observing animal welfare. 388 Laboratory Animals 52(4)

Figure 3. Cage dividers decreased aggressive behavior in light and dark cycles. Divided cages had significantly fewer aggressive behavior events than the standard cages, in terms of daily combined number of events, events in the light cycle, and events in the dark cycle. For daily combined events, light cycle events, and dark cycle events, the number of aggressive behavior events on day 2 were significantly greater than on day 1 and day 7 (post-hoc, y). *p < 0.05 as indicated.

types were excluded from analysis. Comparisons Cage dividers decreased aggressive between types were not conducted. There were no inter- behavior over days and time actions regarding the total number of posturing events between divided and standard cages over time The number of aggressive behavior events significantly (F(2,8) ¼ 2.632, p ¼ 0.132). differed over the time of day on day 1 (F(12,48) ¼ 8.982, p < 0.001; Figure 4(a)), day 2 (F(12,48) ¼ 96.437, Anecdotal findings of divided cages p < 0.001; Figure 4(b)), and day 7 (F(12,48) ¼ 7.017, p < 0.001; Figure 4(c)). Significant interactions between In divided cages, mice spent time in all three burrows the daily combined number of events between divided and the common area, at times alone and together. and standard cages over hours were observed on day 1 Spontaneously, single animals would move into an (F(12,48) ¼ 2.143, p < 0.05) and day 2 (F(12,48) empty burrow without provocation. The nestlet was ¼ 12.429, p < 0.001). Given the largest effect sizes brought into a single burrow and formed into a nest, were observed on day 2, differences between divided where the mice would sleep together. In the standard and standard cages were explored. Significant reduc- cage, the dominant mouse showed activity by repeat- tions in aggressive behavior events occurred in divided edly returning to the nesting area, where the subordin- compared to standard cages on day 2, with significance ate mice slept. Aggressive behaviors in the standard achieved for several hours during the light cycle and cage involved chasing around the circumference of the after dark onset (Figure 4(c)). cage. In the divided cage, escape to or from a burrow stopped the aggressive behavior. Cage dividers decreased the incidence of all aggressive behavior types recorded Discussion Divided cages had significantly fewer aggressive behav- The cage dividers presented here provide a possible ior events than standard cages, in terms of posturing solution to mitigate the aggression that can emerge (F(1,4) ¼ 44.330, p < 0.01), scuffling (F(1,4) ¼ 20.360, from the artificial, open-space, laboratory mouse hous- p < 0.05), and unprovoked biting (F(1,4) ¼ 23.710, ing conditions, which prevent establishing a functional p < 0.01) (Figure 5). For the 12 h observation period dominance hierarchy. Cage dividers were designed to on day 2, more posturing was tallied compared to day 1 emulate the natural environment of wild mice by par- and day 7 (F(2,8) ¼ 24.301, p < 0.001). No events of titioning off a common area and separated ‘‘burrows’’ blood drawn were observed and no veterinarian inter- without compromising cage area or permanently mod- vention was necessary, and therefore these aggression ifying existing caging.26,37 The addition of cage dividers Tallent et al. 389

Figure 4. Cage dividers decreased aggressive behavior over days and time. Diurnal distribution of aggressive behavior following receipt of animals and introduction to new environment over 12 h of day 1 (a), following establishment of the cage hierarchy over 12 h of day 2 (b), and following full acclimation to the cage environment and establishment of a social hierarchy over 12 h of day 7 (c). The number of aggressive behavior events significantly differs over the time of day at day 1, day 2, and day 7. Divided cages significantly reduced aggressive behavior events on day 2, with significance at individual hours shown (post-hoc, y). Significant interactions regarding the daily combined number of events between divided and non-divided cages over hours were observed on day 1 and day 2. *p < 0.05 as indicated. to existing mouse cages significantly attenuated aggres- on day 2 during establishment of the cage hierarchy. sive behavior in group-housed Balb/c male mice. Results from this study indicate partial cage dividers These beneficial effects of cage dividers were observed may be a solution to a pervasive welfare issue for across light cycles, within hours of a single day, research animals, with potential downstream benefits over days of housing, and in terms of aggression type. on research quality. The addition of cage dividers decreased aggressive In this initial, empirical investigation into feasibility posturing, scuffling, and biting behaviors, particularly and efficacy, we report mouse home-cage behavior over 390 Laboratory Animals 52(4)

dividers may provide a more natural environment, i.e. burrows,19,26,37,39 routes of retreat,26,37 separate animalstobreakeyecontact,andleadtoincreased exploration and ambulation.16 Furthermore, the cage dividers did not impede husbandry workflow or daily census and welfare checks.16 The specific features of the divided cage that reduce aggression remains to be determined. Aggressive behaviors can culminate in injury (evidence of blood or need for veterinarian intervention). Intervention typically results in separation and isolation of the animals. As mice are a social species, Figure 5. Cage dividers decreased the incidence of all isolation may result in stress without resolution of atyp- aggressive behavior types recorded. Divided cages have ical behavior.15–17 Continuous stress can affect mouse significantly fewer aggressive behavior events than the physiology in terms of stress hormone regulation and standard cages, in terms of posturing, scuffling, and signaling from wound healing. Veterinary intervention unprovoked biting. On day 2, significantly more posturing can be costly, atypical behavior and physiological y was tallied in comparison to day 1 and day 7 ( ). changes can affect scientific rigor,20,41 and individual Comparisons between types were not conducted. There housing increases per diem costs and cage space avail- were no interactions regarding the total number of posturing events between divided and standard cages over ability. Hence, the downstream impact on research is time. *p < 0.05 as indicated. significant. A reduction in aggressive behavior is indi- cative of a healthy deme hierarchy and reduced stress; both improving research reproducibility and reducing one week, in response to partially divided cages upon technician and veterinarian time. receipt at the animal facility. Day 1 observations rep- Limitations of the present study guide future resented receipt and housing of animals following research. Cage density was pre-selected by the number transport and their introduction to the new environ- of burrows available, and not by industry standards. ment. During this initial period, animals are stressed Cage densities of 4 and 5 mice of various from shipping and handling, and explore the new hous- strains would extend the effectiveness of the partial ing conditions. Day 2 observations represented the cage divider for widespread use.6,16,39 Here we report establishment of the deme hierarchy as evidenced by results over 1 week of housing, where the impact of the highest tallies of aggressive behavior. Day 7 was cage dividers on long-term housing and/or breeding one week after acclimation to the environment with colonies remains to be determined. Further, the current the establishment of a hierarchy, where aggressive design included solid barriers between burrows, creat- events likely have reduced to those necessary to main- ing a ‘‘dead-end’’ if a subordinate were chased. Chasing tain the deme hierarchy. events where an escape hole may have benefited a Animal health is a key component of animal wel- subordinate were not observed. Cage dividers could fare, as is stress and stress management. Territorial or be considered as an intervention instead of isolation resource disputes initiate stress,3–5,12,15,16,19,39 which is for mice that show aggressive behavior. Ongoing evident in decreased exploration and ambula- studies are evaluating the impact of cage dividers on tion.15,16,39 Stress then leads to aggressive behavior behavioral performance in standard neurological in mice,6,7,15–18,20,38,39 and subsequent atypical behav- tasks. Future studies are necessary to investigate iors can be misinterpreted by other mice.3–6,12,38 The corticosterone levels to validate that the addition of dominant animal’s aggressive behavior increases when cage dividers alleviate or minimize stress. hierarchal cues are not responded to appropri- Continued evaluation of partial cage dividers has a ately.3,6,7,15,16,38,39 In a standard cage, subordinate primary benefit towards animal welfare. Results will males cannot avoid line-of-sight of the dominant lead to a better understanding of mice in their home male,7,38 thereby perpetuating a cycle of aggression cage environment, and suggest procedures involving and stress. Environmental enrichment partially cage additions that reduce aggressive behavior and addresses these issues, perhaps by providing a distrac- improve overall welfare. The addition of cage dividers tion, but causes other issues related to limited that create a natural burrow-like environment may resources.16,18–21 Individual animals may be territorial provide more physical activity,19,26,37 improve animal over enrichment objects or perch on top for aggressive welfare by reducing aggressive behavior, and enhance attacks. Complex cages permit species-typical behav- research reproducibility by constraining the influence of ior and provide routes for retreat.26,37 The cage deme hierarchy and abnormal behavior. Tallent et al. 391

Acknowledgements Influence of hierarchy, kinship, and familiarity. J Appl The authors thank Daniel Griffiths for his assistance with Anim Welfare Sci 2001; 4: 91–103. recording equipment. 8. Grant EC and Mackintosh JH. A comparison of the social posture of some common laboratory rodents. Behaviour 1963; 21: 246–259. Declaration of Conflicting Interests 9. Crowcroft P. Mice all over. London: G. T. Foulis, 1966. The author(s) declared the following potential conflicts of 10. Gaskill BN, Stottler AM, Garner JP, et al. The effect of interest with respect to the research, authorship, and/or pub- early life experience, environment, and genetic factors on lication of this article: BRT and JL are inventors on a patent spontaneous home-cage aggression-related wounding in submission for vertebrate animal cage dividers, which is held male C57BL/6 mice. Lab Anim 2017; 46: 176–184. by the University of Arizona. The University of Arizona has a 11. National Research Council. Guide for the care and use of licensing agreement for the manufacture and sales of cage laboratory animals. 8th ed. Washington, DC: National dividers to Helm Technologies, LLC, in which BRT and JL Academies Press, 2010. hold equity positions. 12. Willis-Owen SA and Flint J. The genetic basis of emotional behaviour in mice. Eur J Human Genet 2006; 14: Funding 721–728. The author(s) disclosed receipt of the following financial sup- 13. Wang F, Zhu J, Zhu H, et al. Bidirectional control of port for the research, authorship, and/or publication of this social hierarchy by synaptic efficacy in medial prefrontal article: Studies were funded by Mission Support funds in the cortex. Science 2011; 334: 693–697. Department of Child Health, University of Arizona College 14. Greenberg GD, Howerton CL and Trainor BC. Fighting of Medicine-Phoenix. in the home cage: agonistic encounters and effects on neurobiological markers within the social decision- making network of house mice (Mus musculus). Note Neurosci Lett 2014; 566: 151–155. 15. Moberg GP and Mench JA. The biology of animal stress: These data were reported at the 67th Annual AALAS basic principles and implications for animal welfare. Meeting in Charlotte, North Carolina (Tallent BR and Wallingford, UK: CABI Publishing, 2000. Lifshitz J. Reducing aggression in mice with addition of 16. Van Loo PL, Van Zutphen LF and Baumans V. Male cage dividers. Abstracts of Scientific Presentations 2016 management: coping with aggression problems in male AALAS National Meeting Charlotte, North Carolina. laboratory mice. Lab Anim 2003; 37: 300–313. JAALAS 2016; 55: 606–710). 17. National Research Council. Recognition and alleviation of pain and distress in laboratory animals. Washington, DC: National Academies Press, 1992, p.160. ORCID iD 18. Ambrose N and Morton DB. The use of cage enrichment Jonathan Lifshitz http://orcid.org/0000-0002-4398-6493 to reduce male mouse aggression. J Appl Anim Welfare Sci 2000; 3: 117–125. References 19. Chamove AS. Environmental enrichment: a review. Anim 1. Gray SJ, Jensen SP and Hurst JL. Effects of resource Tech 1989; 40: 155–178. distribution on activity and territory defence in house 20. Van de Weerd HA, Aarsen EL, Mulder A, et al. Effects of mice, Mus domesticus. Anim Behav 2002; 63: 531–539. environmental enrichment for mice: variation in experi- 2. Latham N and Mason G. From house mouse to mouse mental results. J Appl Anim Welfare Sci 2002; 5: 87–109. house: the behavioural biology of free-living Mus muscu- 21. Van de Weerd HA, Van Loo PL, Van Zutphen LF, et al. lus and its implications in the laboratory. Appl Anim Preferences for nesting material as environmental enrich- Behav Sci 2004; 86: 261–289. ment for laboratory mice. Lab Anim 1997; 31: 133–143. 3. Hurst JL, Fang J and Barnard C. The role of substrate 22. Arndt SS, Laarakker MC, van Lith HA, et al. Individual odours in maintaining social tolerance between male housing of mice: impact on behaviour and stress house mice, Mus musculus domesticus: relatedness, inci- responses. Physiol Behav 2009; 97: 385–393. dental kinship effects and the establishment of social 23. Gonder JC and Laber K. A renewed look at laboratory status. Anim Behav 1994; 48: 157–167. rodent housing and management. Ilar J 2007; 48: 29–36. 4. Mackintosh JH. Territory formation by laboratory mice. 24. Bartolomucci A, Palanza P, Sacerdote P, et al. Individual Anim Behav 1970; 18: 177–183. housing induces altered immunoendocrine responses to 5. Mackintosh JH. Factors affecting the recognition of ter- psychological stress in male mice. Psychoneuroendocrino ritory boundaries by mice (Mus musculus). Anim Behav 2003; 28: 540–558. 1973; 21: 464–470. 25. Hunt C and Hambly C. Faecal corticosterone concentra- 6. Poole TB and Morgan HDR. Differences in aggressive tions indicate that separately housed male mice are not behaviour between male mice (Mus musculus L.) in colo- more stressed than group housed males. Physiol Behav nies of different sizes. Anim Behav 1973; 21: 788–795. 2006; 87: 519–526. 7. Van Loo PLP, de Groot AC, Van Zutphen BFM, et al. 26. Chamove AS. Cage design reduces emotionality in mice. Do male mice prefer or avoid each other’s company? Lab Anim 1989; 23: 215–219. 392 Laboratory Animals 52(4)

27. Howerton CL, Garner JP and Mench JA. Effects of a 35. Van Loo PLP, Kruitwagen CLJJ, Koolhaas JM, et al. running wheel-igloo enrichment on aggression, hierarchy Influence of cage enrichment on aggressive behaviour linearity, and stereotypy in group-housed male CD-1 and physiological parameters in male mice. Appl Anim (ICR) mice. Appl Anim Behav Sci 2008; 115: 90–103. Behav Sci 2002; 76: 65–81. 28. Gaskill BN, Pritchett-Corning KR, Gordon CJ, et al. 36. Appleby MC. The probability of linearity in hierarchies. Energy reallocation to breeding performance through Anim Behav 1983; 31: 600–608. improved nest building in laboratory mice. PLoS One 37. Adams N and Boice R. Mouse (Mus) burrows: effects of 2013; 8: e74153. age, strain, and domestication. Anim Learn Behav 1981; 29. Gaskill BN, Winnicker C, Garner JP, et al. The naked truth: 9: 140–144. breeding performance in nude mice with and without nesting 38. Poole TB and Morgan HDR. Social and territorial material. Appl Anim Behav Sci 2013; 143: 110–116. behaviour of laboratory mice (Mus musculus L.) in 30. Gaskill BN, Gordon CJ, Pajor EA, et al. Impact of nest- small complex areas. Anim Behav 1976; 24: 476–480. ing material on mouse body temperature and physiology. 39. Van Loo PL, Mol JA, Koolhaas JM, et al. Modulation of Physiol Behav 2013; 110: 87–95. aggression in male mice: influence of group size and cage 31. Bazille PG, Walden SD, Koniar BL, et al. Commercial size. Physiol Behav 2001; 72: 675–683. cotton nesting material as a predisposing factor for con- 40. Blanchard RJ, Parmigiani S, Bjornson C, et al. junctivitis in athymic nude mice. Lab Anim 2001; 30: Antipredator behavior of Swiss-Webster mice in a visible 40–42. burrow system. Aggressive Behav 1995; 21: 123–136. 32. Swetter BJ, Karpiak CP and Cannon JT. Separating the 41. Kilkenny C, Browne WJ, Cuthill IC, et al. Improving effects of shelter from additional cage enhancements for bioscience research reporting: the ARRIVE guidelines group-housed BALB/cJ mice. Neurosci Lett 2011; 495: for reporting animal research. Osteoarthritis Cartilage 205–209. 2012; 20: 256–260. 33. Van de Weerd HA, Baumans V, Koolhaas JM, et al. 42. Walker MD and Mason G. Female C57BL/6 mice show Strain-specific behavioral-response to environmental consistent individual differences in spontaneous inter- enrichment in the mouse. J Exp Anim Sci 1994; 36: action with environmental enrichment that are predicted 117–127. by neophobia. Behav Brain Res 2011; 224: 207–212. 34. Tischkau SA and Mukai M. Activation of aryl hydrocar- 43. Grossen NE and Kelley MJ. Species-specific behavior bon receptor signaling by cotton balls used for environ- and acquisition of avoidance behavior in rats. J Comp mental enrichment. J Am Assoc Lab Anim 2009; 48: Physiol Psychol 1972; 81: 307–310. 357–362.

Re´sume´ L’agressivite´ chez la souris entraıˆne souvent des blessures conduisant a` des euthanasies non planifieés ou a` l’initiation de protocoles d’isolation des animaux, ce qui augmente les couˆts de recherche et e´puise les ressources. Nous avons cherche´ ici a` savoir si l’ajout d’un se´parateur partiel de cage dans les cages exis- tantes de souris affectait le comportement agressif des souris maˆles logeés en groupe (18 souris; 3 par cage). Les souris ont e´te´ assigneés au hasard a` l’un des deux groupes de`s leur arriveé au vivarium: 1) cage standard; 2) cage doteé d’un se´parateur partiel de cage. L’observation du comportement sur une pe´riode de 12 heures a e´te´ effectueé aux jours un, deux et sept apre`srećeption des souris a` l’installation afin d’e´valuer leur agressivite´ au cours de l’e´tablissement des hie´rarchies de dominance. Les observateurs, ignorant la conception de l’e´tude et son hypothe`se, ont note´ chaque videó selon le nombre et le type de comportements agressifs, qui ont e´te´ additionne´s pour chaque heure et analyse´s. Les re´sultats ont indique´ une diminution statistiquement significative des comportements agressifs des souris logeés dans des cages avec se´parateurs par rapport aux souris logeés dans des cages standard. Nous en concluons que les se´parateurs de cage, qui ressemblent a` des terriers et fournissent l’acce`sa` l’alimentation/l’eau communes, peuvent pro- mouvoir une recherche rigoureuse en re´duisant le nombre d’animaux utilise´s dans une e´tude et en raffinant le logement, tout en ame´liorant le bien-eˆtre des animaux.

Abstract Aggressionen unter Maüsen verursachen haüfig Verletzungen, die zu ungeplanter Einschla¨ferung oder Umsetzung von Protokollen zur Isolierung von Tieren fu¨hren, wodurch Forschungskosten steigen und Ressourcen strapaziert werden. Mit der vorliegenden Studie haben wir getestet, ob durch Hinzufu¨gen eines partiellen Ka¨figteilers in vorhandene Maüseka¨fige aggressives Verhalten bei in Gruppen untergebrach- ten ma¨nnlichen Maüsen (18 Maüse – 3 pro Ka¨fig) beeinflusst wird. Die Maüse wurden bei Ankunft im Gehege zufa¨llig einer von zwei Gruppen zugeordnet: 1. im Standardka¨fig, 2. im Ka¨fig mit partiellem Ka¨figteiler. Verhaltensbeobachtungen u¨ber 12 Stunden wurden am ersten, zweiten und siebten Tag nach Ankunft in Tallent et al. 393

der Einrichtung durchgefu¨hrt, um Aggressionsverhalten im Zuge der Etablierung von Dominanzhierarchien zu bewerten. Beobachter, die zum Testen von Design und Hypothese verblindet wurden, bewerteten die einzel- nen Videos nach Anzahl und Art der aggressiven Verhaltensweisen, die fu¨r jede Stunde aufsummiert und analysiert wurden. Im Ergebnis zeigte sich eine statistisch signifikante Abnahme des aggressiven Verhaltens von Ma¨usen in Ka¨figen mit Trennwa¨nden im Vergleich zu Ma¨usen in Standardka¨figen. Wir kommen daher zu dem Schluss, dass Ka¨figtrennwa¨nde, die an Tierbaue erinnern und Zugang zu gemeinsamem Futter/Wasser ermo¨glichen, intensive Forschung mit dem Ziel fo¨rdern ko¨nnen, die Zahl der in einer Studie verwendeten Tiere zu verringern und die Unterbringung und damit den Tierschutz zu verbessern.

Resumen Las agresiones en ratones a menudo conllevan lesiones que llevan a una eutanasia no planificada o a la iniciacioń de protocolos de aislamiento de animales, por tanto aumentando los costes de investigaciońy limitando los recursos. En este estudio hemos probado a anãdir un separador parcial en las jaulas de ratones existentes para ver si esto tenıá alguń efecto en el comportamiento agresivo en ratones macho enjaulados en grupo (18 ratones; 3 por jaula). Los ratones fueron repartidos aleatoriamente en dos grupos al llegar al vivario: 1) jaula estańdar; 2) jaula con un separador parcial. Se llevo´ a cabo una observacioń del comportamiento durante 12 horas el dıá 1, 2 y 7 tras llegar a las instalaciones a fin de poder evaluar la agresividad durante el periodo de creacioń de jerarquıás de dominancia. Los observadores cegados para estudiar el disenõ y las hipo´tesis puntuaron cada vı´deo seguńelnu´mero y tipo de comportamiento agresivo, los cuales fueron analizados y ponderados cada hora. Los resultados indicaron un descenso estadı´sticamente significativo de los comportamientos agresivos en ratones enjaulados con separadores en comparacioń a los ratones en jaulas estańdar. Podemos concluir que los separadores de jaulas, que se parecen a madrigueras y que dan acceso a agua/comida, pueden fomentar una investigacioń rigurosa reduciendo el nu´mero de animales utilizado en un estudio y refinando el enjaulamiento, con lo cual se mejora el bienestar animal. laboratory an imals Original Article limited Laboratory Animals 2018, Vol. 52(4) 394–401 ! The Author(s) 2018 A longitudinal study on timing and Reprints and permissions: sagepub.co.uk/ velocity of rat molar eruption: journalsPermissions.nav DOI: 10.1177/0023677217750410 Timing of rat molar eruption journals.sagepub.com/home/lan

Balazs J Denes, Aikaterini Lagou, Domna Dorotheou and Stavros Kiliaridis

Abstract Rat molar eruption and occlusion data were compiled from several studies but several inconsistencies were found, rendering the planning of eruptional studies difficult and imprecise. Our aim was to measure eruption and occlusion days, as well as eruption velocity, in the upper and lower three molars from infancy to end of adolescence in the rat. A total of 19 male and female Wistar rats were scanned daily by micro-computed tomography (CT) from day 15 to 70. We measured the eruption of all maxillary and mandibular molars with reference points at the hard palate and mandibular canal at three stages: pre-emergent, pre-occlusal, and functional. Statistical analysis was performed with a mixed-model analysis of variance (ANOVA) and a Sidak post hoc test. The first molar erupts on average on day 17, the second molar on day 20, and the third molar on day 33. The eruption velocity of the first molar was the highest at 90.9 microns/day (standard error (se) ¼ 12.80), followed by the second molar at 65.9 microns/day (se ¼ 5.80), and the lowest was the third at 47.0 microns/day (se ¼ 3.28), (p < 0.001). On average, the pre-occlusal phase had the highest velocity at 97.2 microns/day (se ¼ 1.72), the pre-emergent was lower at 84.9 (se ¼ 2.29), and the functional was the lowest at 21.7 (se ¼ 0.45), (p < 0.001). The eruption rate decreased from the first to third molar and was also different between phases: the pre-occlusal phase had the highest rate, closely followed by the pre-emergent phase while the functional eruption rate was significantly lower than the other phases.

Keywords pre-occlusal eruption, micro-CT, rat molar, velocity, occlusion

Date received: 12 July 2017; accepted: 4 December 2017

Introduction actors in the post-emergent process, further research Tooth eruption is a complex process involving many is required. structures, cellular types, and signaling pathways. Its The albino rat is a well-adapted model for studying physiology has been the subject of research, primarily the eruption process, in particular the rat molars, which focusing on the pre-emergent stage of the eruption pro- are sufficiently similar to human molars to provide cess where the role of the dental follicle, of the alveolar valuable insight. In order to examine their post- bone and of the germ have been well-described.1,2 At emergent stage, one must know the eruption timing of this time, the post-emergent eruption mechanism has rat molars. Hoffman and Schour7,8 published two not been elucidated and the current knowledge relies mostly on hypotheses. The most common are: blood Division d’Orthodontie, Clinique Universitaire de Me´decine flow pressure at the apex, collagen fibers of the peri- Dentaire, Universite´ de Gene`ve, Switzerland odontal ligament, root elongation, pulp and dentin proliferation, rate of alveolar bone remodeling, and Corresponding author: 3–6 Balazs J Denes, Division d’Orthodontie, Clinique Universitaire de presence of occlusal forces. It is possible that one Me´decine Dentaire, Faculte´ de Me´decine, Universite´ de Gene`ve, 19 or more of these factors contribute to the eruption pro- rue Lombard, 1205, Geneva, Switzerland. cess and thus to discover the cellular and molecular Email: [email protected] Denes et al. 395 hallmark studies on rat molar development from birth To examine the influence of the anesthesia procedure to 500 days, which included a detailed table of molar on the well-being of the animals, the weight of the development stages and time. However, these data are control rats was compared with that of the male rats compiled from four different studies and carry the of the experimental group. Furthermore, to ensure biases linked to cross-sectional studies, as well as that that the frequent anesthetic procedure had no adverse of combining results of different origin. For example, effects on tooth eruption, the tooth development stage the day of eruption of the first molar is cited as day 19 of the four first molars was compared between the but Grier and Wise9 observed it to be day 17 and later control and experimental groups. Wise et al.10 cited it as day 18. Although some data on the timing of rat molars exist, inconsistencies between publications indicates the need for a more complete Material and methods investigation, especially whether the mechanism of the Micro-CT pre-occlusal eruption should be investigated. Our goal was to follow the eruption of the three molars of the The Perkin ElmerÕ Quantum GX in vivo micro-CT was Wistar rat in a longitudinal design by the means of used with isoflurane anesthetic gas. The gas was admin- in vivo micro-computed tomography (CT) scans from istered at 5% and 1 l/min for the induction phase and at 15 to 70 days of age to investigate the mean eruption 2% and 1 l/min during the scan. Up to the age of 32 age, occlusion age, and eruption velocities, as well as days, the rats were scanned using the mouse-size tray at the amount of variation in the three molars. 30-micron resolution (standard) and 2 min exposure time. From 34 to 70 days, the rat-size tray was neces- Animals sary and the scanning parameters were adjusted accordingly to maintain resolution: 30-micron (high This study was approved by the ethical committee of resolution) and 4 min exposure time. the Republic and Canton of Geneva, Switzerland under the number GE/72/15. The experimental Eruption group consisted of 19 15-day old rats (8 females and 11 males) of the Rattus norvegicus species, and Wistar The results were exported to OsirixÕ image analysis strain. They were born in-house of two dams with no software. The position of the 12 molars of each rat specific microbiological or immune status or particular was measured on coronal plane slices. To measure the genetic background. The dams were obtained from amount of eruption, we used stable landmarks. In the Janvier Labs, France. All animals were housed in mandible we used the mandibular canal and in the max- the animal facility of the University of Geneva, illa, the roof of the palate above the follicular sac Switzerland and cared for by the laboratory techni- (Figure 1). The outline of the canal and of the follicular cians of the facility and our lab. The experimental sac were traced and the tip of the palatal cusp marked. rats were kept with their dam until weaning on day In the maxilla, the tracing was copied to subsequent 21 and then transferred to new cages of four rats/cage days and positioned to the roof of the alveolar process. and separated by sex. The tooth position was measured between the center of Wistar rats were chosen because they are a widely the tracing and the tip of the cusp. The daily eruption used experimental strain and thanks to the non- velocity was obtained by calculating the difference of invasive in vivo micro-CT method we were able to tooth position between two subsequent measurements maintain a low number of animals. The animals and dividing by the number of days in between. In add- were followed longitudinally with daily in vivo ition, we identified the day the tooth pierced the gingiva micro-CT scans from 15 to 42 post-natal days and (start of pre-occlusal phase) and the first occlusal three times per week from 43 to 70 days. The rats contact (start of functional phase) and used this were marked by piercing of the ears and weighed information to determine the three phases of eruption: three times per week to monitor growth. At the age pre-emergent, pre-occlusal and functional. The begin- of 70 days all the rats were humanely killed by CO2 ning of the pre-occlusal phase was determined by the and the dams were kept for further breeding. One of day the tooth pierced the gingiva. This measurement the rats died in respiratory failure during the scan at was validated to intra-oral photos of the rats ranging the age of 56 days. To control for any adverse effect of from 16 to 28 days to verify accuracy of the method. the repeated micro-CT procedure, the experimental Occlusal contact was determined by at least one group was compared with that of a control group cusp-to-cusp contact between antagonist molars. containing nine male rats. The control group received The opening of the jaws through muscle relaxation a single micro-CT scan, five male rats at the age of 18 during anesthesia was prevented with a tight elastic days and four male rats at the age of 28 days. attached around the snout. 396 Laboratory Animals 52(4) Statistics ANOVA by rearranging the molar and jaw variables For all statistical analyses SPSS Statistics software for into one dummy variable. The tooth position was Windows, version 24.0 (IBM Corp., Armonk, NY, tested using ANOVA with variables sex[F/M], USA) was used. The eruption and occlusion days side[L/R], molar[1,2,3] and a Sidak post hoc test. were analyzed using analysis of variance (ANOVA) The eruption velocity was tested with a mixed-model with the variables sex[F/M], side[L/R], jaw[U/L] ANOVA with the variables sex[F/M], side[L/R], (only for eruption) and molar[1,2,3], and the levels of molar[1,2,3], jaw[U/L] and eruption_stage[pre-emer- the latter were compared with a Sidak post hoc test. We gent, pre-occlusal, functional] and a Sidak post hoc also performed a Pearson’s correlation test between the test was used. weight and the day of eruption as well as weight and occlusion day. Interaction effects between the levels of Error of the method different variables were determined with a one-way The weight of experimental rats was compared with controls with a two-way ANOVA. Cohen’s Kappa statistic was run to determine if there was agreement between intra-oral photos and micro-CT images on the eruption stage of the teeth (i.e., pre-emergent or post-emergent). To assess the error of the method of the occlusion day, the measurement was repeated twice with a 1-week interval. The systematic error was calculated with a paired t-test on the repeated measures, the random error with the Dahlberg formula and a coefficient of correlation was also performed.11

Results Evaluation of errors in the experimental model We found no difference in the average weight of control group compared with the experimental group Figure 1 Illustration of measurements performed to cal- (p ¼ 0.434) and no interaction between the weight and culate to position of the upper and lower jaws, example of the two time-points (p ¼ 0.265) (Table 1(a)). We also the first molars. Tracing of the mandibular canal and the found no difference in the vertical position of the first maxillary follicular sac and dots corresponding to the tip of the cusps are in black and white. The tracing of the fol- molar between the control and experimental groups licular sac was performed on pre-emergent stage molars. (p ¼ 0.805) and no interaction between molar position The distance between the dot and the center of the tracings and the two time-points (p ¼ 0.623) (Table 1(b)). was measured to record to position and calculate eruption The measurement method for the first appearance of velocity. the tooth in the oral cavity (time of eruption) was

Table 1. Mean weight and first molar position of control compared with experimental rats at 18 and 28 days of age. Control and experimental groups were compared with two-way analysis of variance.

Control Experimental

Day mean SD mean SD

(a) Mean weight of animals in grams. 18 39.1 2.3 36.4 2.0 28 75.6 4.0 76.1 4.9

(b) Mean vertical position of first molar, referenced to the hard palate and mandibular canal in microns. 18 2373.7 779.4 2424.2 683.7 28 2951.9 760.5 2800.3 736.6

SD: standard deviation Denes et al. 397

Table 2. Average age of eruption, age of occlusion, and duration of pre-occlusal eruption period in males and females.

Male Female

Molar mean SD mean SD p-value

(a): Time of eruption (days). First 17.7 0.73 17.3 0.77 0.267 Second 20.1 0.55 20.2 0.85 0.853 Third 33.6 0.73 33.3 0.99 0.922

(b): Time of occlusion (days). First 18.5 0.67 18.4 0.50 0.951 Second 23.0 0.65 22.7 0.79 0.902 Third 39.0 1.09 38.8 1.13 0.795

(c): Duration of pre-occlusal eruption (days). First 0.8 1.11 1.1 0.76 0.756 Second 2.9 0.74 2.5 0.87 0.864 Third 5.4 1.15 5.5 1.53 0.746

SD: standard deviation validated by the Cohen’s Kappa test, which showed a Concerning the duration of the pre-occlusal eruption, strong agreement between the clinical situation (intra- the first molar had the shortest eruption period, the oral photo as the gold standard) and the micro-CT second was approximately two- to three-times longer analysis, k ¼ 0.885 (95% confidence interval (CI), than the first molar and the third approximately five- 0.785 to 0.963), p < 0.001. to six-times longer than the first molar (Table 2(c)). Concerning the occlusion day identification, no sys- The ANOVA analysis and Sidak post hoc test revealed tematic error was detected (p ¼ 0.657), the random a significant difference between all three molars error was 0.14 days and the coefficient of correlation (p < 0.001) but no differences between sex and side. was 0.999. Tooth eruption velocity Eruption and occlusion age The eruption velocity of the first molar was the highest On average, the first molar erupted on day 17, the second at 90.9 microns/day (standard error (se) ¼ 12.80), fol- on day 20, and the third on day 33 (Table 2(a)). The lowed by the second molar at 65.9 microns/day ANOVA analysis of the eruption day showed a signifi- (se ¼ 5.80) and the lowest was the third at 47.0 cant effect of the variable molar (p < 0.001) and an inter- microns/day (se ¼ 3.28); all differences were statistically action between molar*jaw (p < 0.001). The Sidak post significant (p < 0.001). Averaging the three molars, the hoc test of the variable molar showed highly significant pre-occlusal phase had the highest velocity at 97.2 differences between all three molars (p < 0.001). The microns/day (se ¼ 1.72), the pre-emergent was 84.9 interaction between molar and jaw was significant only (se ¼ 2.29) and the functional was the lowest at 21.7 for the third molar (p ¼ 0.001) where the maxillary (se ¼ 0.45). The differences between the three phases molars erupted 0.7 days after the mandibular. No stat- were statistically significant (p < 0.001). istically significant correlation was found between the The mixed-model ANOVA of the eruption velocity eruption day and the weight of the animals. showed a significant effect of eruption_stage (p < 0.001) On average, the occlusion age for the first molar was and interactions with sex (p ¼ 0.026) and molar day 19, the second molar was day 23 and the third was (p < 0.001). The interaction with the variable sex was day 39 (Table 2(b)). The ANOVA analysis showed a statistically significant in the pre-emergent stage significant effect only in the variable molar (p < 0.001) (p ¼ 0.033) and functional stage (p ¼ 0.013) (Table 3(a)). indicating that all molars occluded on different days The interaction with the variable molar was statistically and no interaction between sex, side, and molar.No significant in all three stages (p 0.003). The variables statistically significant correlation was found between molar (p < 0.001) and jaw (p < 0.001) showed statistically the occlusion day and the weight of the animals. significant simple main effects. The mandibular molars 398 Laboratory Animals 52(4)

Table 3. Average eruption velocity and standard deviation of pre-emergent, pre-occlusal, and post-occlusal phases.

Male Female

Eruption stage mean SD mean SD p-value

(a): Eruption velocity at different stages compared between sexes (microns/day). Pre-emergent 80.0 38.5 89.8 51.6 0.033 Pre-occlusal 97.3 33.6 97.1 37.2 0.936 Functional 22.8 9.7 20.6 8.1 0.013 Maxilla Mandible Eruption stage mean SD mean SD p-value

(b): Eruption velocity at different stages compared between jaws (microns/day). Pre-emergent 79.6 46.9 88.6 42.0 0.048 Pre-occlusal 91.4 26.0 103.1 41.6 0.001 Functional 19.6 7.0 24.2 10.4 0.000 First molar Second molar Third molar Eruption stage mean SD mean SD p-value

(c): Eruption velocity at different stages compared between molars (microns/day). Pre-emergent 122.8 50.6 73.1a 31.2 56.5a 9.1 Pre-occlusal 126.7 31.0 95.5 28.6 69.6 17.1 Functional 22.6 5.9 28.6 6.7 14.5 8.4

SD: standard deviation All row-wise p-values p < 0.001 except ap ¼ 0.003.

had a higher eruption rate in all three stages (Table 3(b)). groups, neither in weight nor in tooth eruption. Our The first molar had the highest rate, followed by the findings validate this longitudinal micro-CT approach second molar, and the slowest was the third molar for following tooth eruption in rats as an alternative to (p 0.003) except for the functional phase where the the traditional cross-sectional model. second molar was the fastest (Table 3(c)). The variation of the eruption and occlusion day ranged between 1 day, showing that there was a Discussion high range of variation between animals. For the most part, this variation could not be attributed to The aim of this study was to establish the precise time of the side, jaw, sex, or weight of the animals with the eruption day, occlusion day and eruption velocities of exception of the third molar where the mandibular albino Wistar rat molars by following the eruption lon- molars erupted a half a day sooner than the maxillary gitudinally with in vivo micro-CT scans from 15 to 70 molars. This finding is contrary to Hoffman and days of age. The first molar erupted on day 17 and Schour7 who stated all the maxillary molars to erupt occluded on day 18 with an overall velocity of 90.9 one day later than the mandibular. The eruption pro- microns/day, the second molar erupted on day 20 and cess being a complex multifactorial process, the precise occluded on day 23 with an overall velocity of 65.9 prediction of eruption and occlusion days is not pos- microns/day, and the third molar erupted on day 33 sible with simple factors such as weight or sex. Thus, and occluded on day 39 with an overall velocity of planning of experiments that concern the pre-occlusal 47.0 microns/day. To ensure that the repeated anesthe- eruption must take into account and compensate for sia and micro-CT scanning did not affect the general and this variation either by monitoring the eruption with dental development of the rats, we compared the aver- in vivo micro-CT, increasing the number of animals age weight and vertical tooth position of the experimen- or choosing to study the second or third molar where tal rats with control rats that were not exposed to the the pre-occlusal eruption time is longer. repeated procedure. Our findings indicated no signifi- Although Hoffman and Schour7 have previously cant differences between the control and experimental described the day of eruption and of first occlusion, Denes et al. 399 their data show discrepancies with later studies. The skeletal growth decreases as the animal approaches its eruption day of the first molar is stated as day 19, as adult size and the functional eruption increasingly opposed to Grier and Wise9 who found it to be day 17 serves to compensate for occlusal wear. The functional and Wise et al.10 day 18. Furthermore, the period eruption is a particularly important process to under- between the first molar and second molar eruption stand because it is linked to many pathologies: anterior varies between 3 days after the first molar7 and 7 and lateral open bite, overeruption of unopposed teeth days.10 Our study brings a more precise description of and occlusal height differences with implant supported eruption and occlusion day, including the standard restorations.13–15 The underlying mechanisms of the deviations of the measurements, which tend to indicate post-emergent eruption and their regulators have not that most of the differences between studies probably been identified, but the main tissues involved are the arises from the physiologic variations. Different strains alveolar bone and the periodontal ligament. The alveo- of albino rat could also be an influencing factor but this lar bone remodeling rate might be a limiting factor to hypothesis is not retained because both studies used the eruption through the attachments to the tooth via same Harlan Sprague-Dawley strains.9,10 Another supracrestal collagen fibers,16 but most likely only hypothesis is the variation in environmental conditions well into adulthood when overall growth potential is such as housing and social conditions which can influ- low. The periodontal ligament on other hand may ence tooth eruption through stem cells or epigenetic have the capacity to change the eruption rate through markers. Our results are in line with eruption and signaling pathways in response to local occlusal condi- occlusion days cited by other publications when we tions. There may also be preprogrammed genetic differ- include the physiologic variability. In general, the erup- ences in between the different molars that define the tion times that we observed were shorter than Hoffman overall eruption rate depending on the amount of and Schour7 but the eruption rates were rather similar.8 alveolar bone growth that is necessary in a given The eruption of the first and second molars occurred region. Most likely, both of these mechanisms exist sim- 3 days apart while the third molar erupted approxi- ultaneously, with the genetic component being the mately 2 weeks after the second molar. The duration source of eruption potential and the occlusal stimuli of the pre-occlusal phase showed a linear increase from acting on it as a regulator. the first to the third molar, with an increment of One limitation of this study is that the results apply approximately 2 days, indicating that the eruption only to one strain of rats (Wistar) and therefore the rate must decrease. Indeed, our results show a decrease conclusion cannot be assumed to be exact for other of eruption rate from first to third molars in both the strains, although they can serve as general guidelines. pre-emergent and pre-occlusal phases. The duration of This data improves our knowledge of the eruption eruption is 2–3 times longer in second molar and 5–6 pattern of rat molars and may serve in the planning of times in the third compared with the first molar, how- future studies related to the post-emergent eruption. ever the difference in eruption rate is only 1.3 times and Our current understanding of this mechanism is incom- 1.8 times respectively. This difference between duration plete and needs further investigation and the rat is well- of eruption and eruption velocity between the three adapted model. Furthermore, we have shown that molars can be explained by the continuous eruption repeated in vivo micro-CT scanning is an effective of the adjacent molars, which continue to increase the way to study tooth eruption with a longitudinal design. alveolar bone height and thus increase the eruption distance to the occlusal plane. If this functional eruption is Declaration of Conflicting Interests taken into account, the pre-occlusal eruption velocity The author(s) declared no potential conflicts of interest with difference explains most of the difference observed respect to the research, authorship, and/or publication of this between the three molars in pre-occlusal eruption dur- article. ation. Due to the morphology of the rat jaws, the alveolar height needs to develop taller in the mesial region Funding compared with the distal. In other words, this variation The author(s) disclosed receipt of the following financial of eruption velocity may be a key factor for the control support for the research, authorship, and/or publication of lower face vertical growth, (i.e., the difference of this article: Schweizerischer Nationalfonds zur Fo¨rderung between short- and long-face tendencies).12 der Wissenschaftlichen Forschung, (Grant/Award Number: In the functional phase, the differences between the ‘31003A_144202’). molars is smaller and the second molar has the highest rate. The teeth remain in occlusion during the func- References tional period by means of the functional eruption, 1. Cahill DR and Marks SC. Tooth eruption: Evidence for which is necessary for compensating the vertical skel- the central role of the dental follicle. J Oral Pathol Med etal growth and the occlusal wear.12 Progressively the 1980; 9: 189–200. 400 Laboratory Animals 52(4)

2. Marks SJ and Cahill DR. Regional control by the dental 9. Grier RL and Wise GE. Inhibition of tooth eruption in follicle of alterations in alveolar bone metabolism during the rat by a bisphosphonate. J Dent Res 1998; 77: 8–15. tooth eruption. J Oral Pathol Med 1987; 16: 164–169. 10. Wise GE, Yao S and Henk WG. Bone formation as a 3. Proffit W and Frazier-Bowers S. Mechanism and control potential motive force of tooth eruption in the rat molar. of tooth eruption: overview and clinical implications. Clin Anat 2007; 20: 632–639. Orthod Craniofac Res 2009; 12: 59–66. 11. Houston WJB. The analysis of errors in orthodontic 4. Steedle JR and Proffit WR. The pattern and control of measurements. Am J Orthod 1983; 83: 382–390. eruptive tooth movements. Am J Orthod 1985; 87: 56–66. 12. Lee CF and Proffit WR. The daily rhythm of tooth erup- 5. Cheek CC, Paterson RL and Proffit WR. Response of tion. Am J Orthod Dentofacial Orthop 1995; 107: 38–47. erupting human second premolars to blood flow changes. 13. Iseri H and Solow B. Continued eruption of maxillary Arch Oral Biol 2002; 47: 851–858. incisors and first molars in girls from 9 to 25 years, stu- 6. Dorotheou D, Farsadaki V, Bochaton-Piallat M-L, et al. died by the implant method. Eur J Orthod 1996; 18: Increased cell proliferation and gene expression of genes 245–256. related to bone remodeling, cell adhesion and collagen 14. Tanaka S. Morphological study of open bite. Skeletal metabolism in the periodontal ligament of unopposed Class I and Class II open bite. Aichi Gakuin Daigaku molars in growing rats. Front Physiol 2017; 10(8): 75. Shigakkai Shi 1990; 28: 1129–1150. doi: 10.3389/fphys.2017.00075. 15. Bernard JP, Schatz JP, Christou P, et al. Long-term ver- 7. Hoffman MM and Schour I. Quantitative studies in the tical changes of the anterior maxillary teeth adjacent to development of the rat molar. I. The growth pattern of the single implants in young and mature adults. J Clin primary and secondary dentin (from birth to 500 days of Periodontol 2004; 31: 1024–1028. age). Anat Rec 1940; 78: 233–251. 16. Ingber JS. Forced Eruption: Part I. A method of treating 8. Hoffman MM and Schour I. Quantitative studies in the isolated one and two wall infrabony osseous defects – development of the rat molar II. Alveolar bone, cementum rationale and case report. J Periodontol 1974; 45: and eruption (from birth to 500 days). Am J Orthod Oral 199–206. Surg 1940; 26: 854–874.

Re´sume´ Les donneés sur l’e´ruption des molaires et l’occlusion chez le rat ont e´te´ compileés a` partir de plusieurs e´tudes mais plusieurs incohe´rences ont e´te´ trouveés, ce qui rend la planification des e´tudes sur l’e´ruption difficile et imprećise. Notre objectif e´tait de mesurer les jours d’e´ruption et d’occlusion, ainsi que la vitesse d’e´ruption, au niveau des trois molaires supe´rieures et infe´rieures de l’enfance a` la fin de l’adolescence chez le rat. Dix-neuf rats Wistar maˆles et femelles ont e´te´ scanne´s quotidiennement par microtomographie aux rayons X du jour 15 au jour 70. Nous avons mesure´ l’e´ruption de toutes les molaires maxillaires et mandi- bulaires avec des points de re´fe´rence au niveau du palais dur et du canal mandibulaire a` trois e´tapes: avant l’e´mergence, avant l’occlusion et a` l’e´tape fonctionnelle. L’analyse statistique a e´te´ reáliseé avec un mode`le mixte ANOVA et un test post hoc de Sidak. La premie`re molaire sort en moyenne au 17e jour, la deuxie`me molaire au 20e et la troisie`me molaire au 33e. La vitesse d’e´ruption de la premie`re molaire infe´rieure e´tait la plus e´leveéa` 90.9 microns/jour (ET ¼ 12.80), suivie de la deuxie`me molaire infe´rieure a` 65.9 microns/jour (ET ¼ 5.80) et la plus basse e´tait la troisie`me a` 47.0 microns/jour (ET ¼ 3.28), (p < 0.001). En moyenne, la phase preócclusale est la plus rapide a` 97.2 microns/jour (ET ¼ 1.72), la phase pre´-e´mergence e´tait infe´rieure a` 84.9 (ET ¼ 2.29) et la phase fonctionnelle e´tait la plus basse a` 21.7 (ET ¼ 0.45), (p < 0.001). Le taux d’e´ruption a diminue´ de la 1e`re a` la 3e molaire et e´tait e´galement diffe´rent entre phases: la phase occlusale pre´sentait le taux le plus e´leve´, suivi de pre`s par la phase de pre´-e´mergence alors que le taux d’e´ruption fonctionnelle s’ave´rait significativement plus faible que les autres phases.

Abstract Bei der Zusammenstellung von Daten zu Molareneruption und -okklusion aus mehreren Studien wurden verschiedene Diskrepanzen festgestellt, weshalb sich die Planung von Eruptionsstudien schwierig und unge- nau gestaltete. Unser Ziel war es, die Eruptions- und Okklusionstage sowie die Eruptionsgeschwindigkeit bei den oberen und unteren drei Molaren vom Infantstadium bis zum Ende der Adoleszenz bei der Ratte zu messen. Dazu wurden neunzehn ma¨nnliche und weibliche Wistar-Ratten von Tag 15 bis 70 ta¨glich mittels Mikro-CT-Scanner untersucht. Wir haben den Ausbruch aller Molaren des Ober- und Unterkiefers mit Referenzpunkten am harten Gaumen und Unterkieferkanal in drei Phasen gemessen: praëmergent, praökk- lusal und funktionell. Die statistische Analyse wurde mit einer gemischten ANOVA und einem Sidak-Post- hoc-Test durchgefu¨hrt. Der erste Molar bricht durchschnittlich am Tag 17 durch, der zweite Molar am Tag 20 Denes et al. 401 und der dritte Molar am Tag 33. Die Eruptionsgeschwindigkeit des ersten Molars war mit 90.9 Mikron/Tag am ho¨chsten (sd ¼ 12.80), betrug beim zweiten Molar 65.9 Mikron/Tag (sd ¼ 5.80) und war beim dritten mit 47.0 Mikron/Tag (sd ¼ 3.28) am niedrigsten, (p < 0.001). Im Durchschnitt wurde in der praökklusalen Phase mit 97.2 Mikron/Tag (sd ¼ 1.72) die ho¨chste Geschwindigkeit festgestellt, die in der praëmergenten Phase war niedriger und betrug 84.9 (sd ¼ 2.29), und die Geschwindigkeit in der funktionellen Phase war mit 21.7 (sd ¼ 0.45) die niedrigste, (p < 0.001). Die Eruptionsrate verringerte sich vom 1. zum 3. Molar und unterschied sich auch zwischen den Phasen: Die praökklusale Phase hatte die ho¨chste Rate, dicht gefolgt von der praëmergenten Phase, wa¨hrend die funktionelle Eruptionsrate signifikant niedriger war als die der anderen Phasen.

Resumen Se recopilaron datos sobre oclusioń y erupcioń molar en ratas a trave´s de varios estudios pero se encontraron varias inconsistencias, lo que hizo que la planificacioń de estudios sobre erupcioń se convirtiera en una tarea difıćil e imprecisa. Nuestro objetivo era medir los dıás de oclusioń y erupcioń, adema´s de la velocidad de la erupcioń, en los tres molares superiores e inferiores desde la infancia hasta el final de la adolescencia en ratas. Se escanearon a diario 19 ratas Wistar macho y hembra mediante micro-CT del dıá 15 al 70. Medimos la erupcioń de todas las molares maxilares y mandibulares con puntos de referencia en el canal mandibular y en el paladar duro en tres fases: pre emergente, pre oclusal y funcional. Se llevo´ a cabo un ana´lisis estadı´stico mediante una prueba post hoc con un modelo mezclado de ANOVA y Sidak. El primer mola erupciona el dıá 17, de promedio, el segundo molar el dıá 20 y el tercero el dıá 33. La velocidad de erupcioń del primer molar fue el ma´s alto con 90.9 micrones/dıá (sd ¼ 12.80), seguido del segundo molar con 65.9 micrones/dıá (sd ¼ 5.80) y el ma´s bajo siendo el tercero con 47.0 micrones/dıá (sd ¼ 3.28), (p < 0.001). De promedio, la fase pre oclusal obtuvo la mayor velocidad a 97.2 micrones/dıá (sd ¼ 1.72), la fase pre emergente fue inferior con 84.9 (sd ¼ 2.29) y la funcional fue la ma´s baja con 21.7 (sd ¼ 0.45), (p < 0.001). El porcentaje de erupcioń bajo del primer al tercer molar y tambień fue diferente entre fases: la pase pre oclusal obtuvo el mayor porcentaje, seguida de cerca por la fase pre emergente mientras que el porcentaje de la erupcioń funcional fue significativamente inferior que las otras dos fases. laboratory an imals Original Article limited Laboratory Animals 2018, Vol. 52(4) 402–412 ! The Author(s) 2018 Creating effective biocontainment facilities Reprints and permissions: sagepub.co.uk/ and maintenance protocols for raising journalsPermissions.nav DOI: 10.1177/0023677217750691 specific pathogen-free, severe combined journals.sagepub.com/home/lan immunodeficient (SCID) pigs

Ellis J Powell1, Sara Charley1, Adeline N Boettcher1, Lisa Varley1, Justin Brown2, Martine Schroyen1, Malavika K Adur1, Susan Dekkers3, Dean Isaacson4, Mary Sauer4, Joan Cunnick1, N Matthew Ellinwood1, Jason W Ross1, Jack CM Dekkers1 and Christopher K Tuggle1

Abstract Severe combined immunodeficiency (SCID) is defined by the lack of an adaptive immune system. Mutations causing SCID are found naturally in humans, mice, horses, dogs, and recently in pigs, with the serendipitous discovery of the Iowa State University SCID pigs. As research models, SCID animals are naturally tolerant of xenotransplantation and offer valuable insight into research areas such as regenerative medicine, cancer therapy, as well as immune cell signaling mechanisms. Large-animal biomedical models, particularly pigs, are increasingly essential to advance the efficacy and safety of novel regenerative therapies on human disease. Thus, there is a need to create practical approaches to maintain hygienic severe immunocompromised porcine models for exploratory medical research. Such research often requires stable genetic lines for replication and survival of healthy SCID animals for months post-treatment. A further hurdle in the development of the ISU SCID pig as a biomedical model involved the establishment of facilities and protocols necessary to obtain clean SPF piglets from the conventional pig farm on which they were discovered. A colony of homozygous SCID boars and SPF carrier sows has been created and maintained through selective breeding, bone marrow transplants, innovative husbandry techniques, and the development of biocontainment facilities.

Keywords severe combined immunodeficiency (SCID), snatch farrow, pig, colostrum, specific pathogen-free (SPF)

Date received: 14 June 2017; accepted: 5 December 2017

Introduction Artemis gene segregate in a Mendelian recessive mode of inheritance in which homozygotes or compound het- The Iowa State University (ISU) severe combined erozygotes produce the SCID phenotype.2 Consistent immunodeficiency (SCID) pigs were unexpectedly discovered during a viral challenge study in collaboration 1 1 Department of Animal Science, Iowa State University, USA with Kansas State University. Animals that died early 2Swine Medicine Education Center, Iowa State University, USA in the trial revealed a complete lack of antibodies and 3Thielen Student Health Center, Iowa State University, USA dysplastic immune tissues (thymus, spleen, and lymph 4Laboratory Animal Resources, Iowa State University, USA nodes). Genetic analysis revealed a significant associ- Corresponding author: ation with the chromosome 10 region containing the Christopher K Tuggle, Department of Animal Science, Iowa State Artemis gene, which is associated with human University, 806 Stange Rd, 2255 Kildee Hall, Ames, IA 50011, USA. 2,3 SCID. Two separate point mutations within the Email: [email protected] Powell et al. 403 with human Artemis SCID patients, early in life the or unvaccinated sow. An interesting study22 compared ISU SCID pig is devoid of B and T cells,4,2 but has a circulating immunoglobulin (Ig)G levels between nat- population of natural killer (NK) cells that demonstrate urally suckling and CD piglets: The CD piglets were fed normal function in vitro.5 either milk replacer, bovine colostrum, or pig IgG. While SCID is naturally occurring in humans, mice, Naturally suckling piglets achieved the highest levels dogs, horses, and now pigs,3,6,7,1 it has also been of total IgG, while supplementation with bovine or introduced into rodent and porcine hosts for use pig IgG was sufficient for 100% survival of piglets. as biomedical models through targeted genetic However, only 30% of milk replacer-alone CD piglets modification.8–12 survived.22 While much of the CD piglet-rearing litera- The ISU SCID pig permits the growth of two kinds ture was developed prior to the availability of SCID of human cancer cell lines when injected into the ear,13 pigs, these results indicate that various options may establishing its value for research in cancer drug testing. be available to rear SCID pigs isolated from their nur- The ISU SCID pig has also been used to explore the sing sow, which is the main source of disease transmis- roles of the innate and adaptive immune system during sion to the piglet. a disease challenge, contributing to research focusing This manuscript describes the facilities and protocols on mechanisms of disease for influenza A.14 Although developed to greatly decrease opportunities for contam- there is widespread interest in developing SCID pigs as ination and raise SPF SCID and non-SCID pigs a research model, little research is available on the care derived from non-SPF sows. This work also provides of large-animal SCID models. examples of facilities and protocols to produce disease- Specific pathogen-free (SPF) SCID mice colonies are naive pigs, which may be important for disease chal- typically cared for in a ‘‘barrier facility’’ with group- lenge studies. The farrowing protocols herein described housing boxes under high-efficiency particulate air provide means for creating founding SPF SCID carrier (HEPA)-filtered conditions.15 Staff interacting with ani- dams, methods to introduce new genetics or replace mals follow strict personal protective equipment (PPE) animals within an established SPF colony, and provide requirements and animals are maintained with auto- recommended guidelines for researchers seeking to claved feed and bedding.16 Large-animal facilities develop a SCID pig model containment facility. have many more parameters to balance considering the size and requirements of pigs. We have been unable to keep the immune-compromised SCID pig Animals alive for more than 80 days in a conventional pig-hold- Institutional Animal Care and Use ing facility or within an isolated standard animal room. Committee (IACUC) statement Similar viability of SCID pigs including failure to thrive phenotypes were described after engineering defective All care and animal procedures were approved by the recombination activating genes (RAG) SCID pigs that ISU IACUC and adhere to the United States had to be terminated before 30 days of age.11 To Department of Agriculture guide to Large Animals improve the research potential of the SCID pig model and the Animal Welfare Act. Euthanasia via captive by producing longer-lived, healthy SCID pigs, develop- bolt or intravenous overdose of sodium pentobarbital ment of biocontainment facilities and appropriate was performed by trained staff. methodologies to raise SPF SCID pigs are needed. Current approaches for obtaining SPF piglets from SCID pigs and non-SCID littermates non-SPF sows include snatch farrowing (SF) or cesarean-derived piglets, of which both may involve colos- Yorkshire SCID carrier dams were raised at the ISU trum deprivation (CD).17 The risks of CD are well Lauren Christian Swine Research Center (LCSRC), a understood, and include mortality, reduced protection non-SPF farm environment (see Table 1). Pregnant from clinical disease, and significantly lower hematocrit SCID carrier dams with potential to produce SCID- and hemoglobin levels compared to naturally sucking affected piglets for biomedical use were farrowed in piglets.18–22 Colostrum consumption is positively asso- facilities with high-sanitation clean rooms at the ISU ciated with piglet immunity, survival, and growth.21,23 College of Veterinary Medicine. Common practices for obtaining SPF piglets from non- SPF sows include supplementing piglets with milk 24 Bone marrow transplantation replacer or a bovine colostrum alternative. The pro- (BMT)-rescued SCID sires duction of dried replacement bovine colostrum is an involved process,25 and does not protect the piglet To create litters composed of 50% SCID piglets, we from porcine-specific pathogens with antibodies they rescued SCID boars via BMT, which resulted in a gen- would normally receive from suckling a vaccinated etically SCID, phenotypically normal animal that could 404 Laboratory Animals 52(4)

Table 1. Health testing for the Lauren Christian Swine Research Center (LCSRC) from 2015 to 2017. Carrier dams are sourced from the LCSRC, where animals are routinely monitored for herd health by testing for the pathogens listed. All testing was performed by the Veterinary Diagnostic Laboratory.

Source farm records Pathogen Type of sample (routine testing 2015–2017

Porcine reproductive and respiratory syndrome Sera, oral fluids, tissue (ELISA, PCR) Negativea Porcine parvovirus hemagglutination inhibition Sera (PCR) Negative Mycoplasma hyopneumoniae Tissue, oral fluid (PCR) Negative Lawsonia intracellularis Fecal, tissue (PCR, IHC) Negative Swine influenza virus NP Sera, oral fluid ELISA, PCR) Positive Actinobacillus pleuropneumoniae Tissue (culture) Positive Culture Staph aureus Tissue (culture) Positive Culture Staph hyicus Tissue (culture) Positive Culture Strep suis Tissue (culture) Positive Porcine epidemic diarrhea whole virus Fecal (ELISA, PCR) Negative Ingezim circovirus Tissue (PCR) Negative Rotavirus A, B, C Fecal, tissue (PCR) Positive aSingle individual ELISA positive out of 30 sera samples in 2015; was not confirmed with PCR. Thought to be a contamination error or a false positive. NP: nucleoprotein; ELISA: enzyme-linked immunosorbent assay; PCR: polymerase chain reaction; IHC: immunohistochemistry. be used for breeding. Since successful BMT boars have mixture aerosolized treatment of the room and any resi- intact immune systems, they can be housed in clean, dent equipment. more traditional pig facilities at lower cost. To accomplish this, SCID piglet-containing litters were major Maintenance and care of animals histocompatibility complex-typed to identify recipient and donor littermate matches.26 SCID recipients SCID carrier dams. Two sources of pregnant dams received unfractionated bone marrow cells (isolated were used in this project: the LCSRC and from from all long bones of the donor) intravenously deliv- within the biocontainment facility. Artificial insemin- ered via an ear vein. Successful engraftment was veri- ation is used at both locations. Before leaving the fied by complete blood cell count, flow cytometry, and LCSRC, pregnant carrier sows are tested by blood response to vaccination. draw for porcine reproductive and respiratory syndrome (PRRS), porcine circovirus (PCV2), and porcine epidemic diarrhea virus (PEDV) through the ISU Materials and methods Veterinary Diagnostic Laboratory (VDL). Test-nega- Maintenance and construction of facility tive sows are washed at the farm, delivered by pre-sani- tized truck and trailer, washed again, and housed in a high-sanitation room with other separately penned Bubble fabrication. Custom design of bubbles gestating sows within the LAR facility. A week before was completed with expertise from ISU, SCID project farrowing, animals are transferred to a recently cleaned leadership, Laboratory Animal Resources (LAR) and Halo’d room containing a farrowing crate, and supervisors, and Biobubble Inc, which created the sows are cleaned with chlorhexidine. Once piglets are final design, and fabricated and installed the main iso- removed (delivered for SF (see below), or weaned for lation equipment. Incoming conditioned city water is naturally farrowing) sows are transferred back to the ultraviolet (UV)-irradiated and filtered (5 micron sow room. If sows are to be used for artificial insemin- before UV irradiation and 0.5 micron after UV irradiation within the biocontainment facility where no ation) (Figure 1(d)). live boar was available, they are monitored for behavioral estrus (standing heat) with a cloth sprayed with Room sterilization. Prior to animal occupation, rooms boar pheromones (Part #: 320506, QC Supply, are sterilized (Halo’d) using a HaloMistTM Disinfectant Schuyler, NE, USA). If natural heats are not detected, Fogging Solution (halosil.com/halo-disinfection- adjusting the estrous cycle in preparation for insemin- system), which entails a hydrogen peroxide/silver ation of sows was accomplished by administering Powell et al. 405

Figure 1. General bubble components. (a) Entrance of long-term bubble; pigs are housed on either side while middle section is designated for dressing in PPE. (b) Staff shown collecting cord blood on newly snatch-farrowed piglets in the STB. (c) HEPA filtration unit filters all incoming air that enters the bubble. (d) Water is filtered and irradiated before entering the bubble. (e) All staff don PPE prior to entering either bubble. PPE: personal protective equipment; STB: short-term bubble; HEPA: high-efficiency particulate air.

15 mg/day altrenogest (Matrix, Merck, NJ, USA) orally pasteurized at the ISU dairy farm using standard for 18 days followed by one dose of PG600 (Merck, Part bovine practices. Confirmation of pasteurization was #: 147448) administered intramuscularly 24 hours after accomplished with a Bulk Tank Analysis/Standard last Matrix dose, and a single dose of Ovugel (JBS Plate Count at the ISU VDL, which includes the United, Sheridan, IN, USA, NDC #: 51233-101-50) detection of Streptococcus agalactiae, Staphylococcus administered intravaginally approximately 80 hours aureus, coliforms, gram-negative rods (lactose nega- after PG600 injection. Behavioral estrus was detected tive), non-agalactiae Streptococci, coagulase-negative typically 24–48 hours post-Ovugel administration, at Staphylococci, Corynebacterium,andBacillus, which point animals are artificially inseminated using Mycoplasma, among others. Pasteurization of colostrum fresh or frozen semen from the desired sire. dramatically decreases, but does not eliminate, microbial counts. For example, one unpasteurized colostrum Neonatal piglet care. Naturally farrowed piglets are sample had colony-forming units (CFU) counts of monitored to ensure they are suckling. SF piglets are 3.9 Â 105 CFU/ml while post-pasteurization averages delivered by personnel wearing PPE (see below) with- 3.8 Â 103 CFU/ml. Piglets were fed with a 60 ml syringe out touching the floor or crate using sterile towels and and attached sterile tubing (Part #: 366.78750.4, Midwest transferred into sterile rodent boxes with air-filtered lids Veterinary Supply, MN, USA) (Figure 2(b)). After (Innovive, Part #: RS1-H) for transport to the short- 250 ml colostrum consumption, piglets are converted to term bubble (STB), where they are dried with Mistral irradiated milk replacer, and eventually starter feed. micronized clay (Part #: 541005, QC Supply, NE, USA), weighed, and placed in heat lamp-warmed, General piglet care. All litters are processed 24–48 piglet-housing decks (BirthrightTM, Yellow Sow, hours post-birth; piglets were ear notched, their needle Ralco, MN, USA). Within the first 24 hours of life, teeth clipped and tails docked, and given 1 ml each of SF piglets were fed a minimum of 250 ml27 pooled iron (Iron Dextran cat# DU3067, Durvet, MO, USA) pig colostrum collected previously by hand by staff at and EXCEDE (ceftiofur crystalline free acid, used to the LCSRC from farrowing sows, which was then manufacturer’s specifications, Zoetis, NJ, USA). 406 Laboratory Animals 52(4)

Piglets are weighed daily until achieving a body weight plastic suits in hallways and new suits are donned of 20 kg, and weekly thereafter up to 100 kg. All piglets before entering a new animal room. Rubber slip-on are weaned from the sow or from milk replacer at 24–35 shoes are worn between animal containing units, and days of age and transitioned to an irradiated starter feed. changed for rubber boots that remain inside pig-resid- All feed given to pigs other than colostrum (gestation, ing areas. starter, and milk replacer powder) is gamma-irradiated (5 kGy, Iotron Inc). Long-term carrier animals are vac- Monitoring bubble and research pig health. All pigs cinated following standard pig farm practices including are checked by staff twice per day, with written records Circumvent PCV-M (Intervet), FarrowSure Gold B updated daily. In accordance with biosecurity manage- (Pfizer), and LitterGuard LT-C (Pfizer). To aid in the ment, staff enter the cleanest space first (usually the safety and efficiency of animal handling, as well as LTB), and as described above change outer suits and increased animal enrichment, pigs in our facility are gloves before entering the STB. Only outer gloves are clicker-trained with marshmallow rewards for regular changed for entry into isolated non-SCID sow rooms care such as weighing, hoof trimming, vaccinations, (or housed naturally farrowed litters). If any animal in snaring, and additional sample collection. any given location is not Bright, Alert, Responsive (BAR) or Quiet, Alert, Responsive (QAR), an on-call IgG enzyme-linked immunosorbent veterinarian is called. Depending on the symptoms pre- assay (ELISA) sent and severity, animals are closely monitored with scheduled veterinarian check-ups, and if necessary, A sandwich ELISA was used to quantify pig serum IgG veterinarian-prescribed treatments. concentration. The protocol, adapted from Bethyl Laboratories Porcine IgG ELISA protocols, used cap- Monitoring LCSRC pig health. Animals are routinely ture antibody of polyclonal goat anti-porcine IgG tested six times a year unless concerns or symptoms (Bethyl Labs #A100-104 A, 10 mg/ml). Unconjugated warrant additional testing for the specific pathogens pig IgG reference serum (Bethyl Labs #RS10-107) listed (Table 1). The number of animals selected for was used as a standard for serially diluted experimental testing was based on the estimated prevalence and a serum samples. Detection antibodies to pig IgG-Fc 95% confidence level that the disease will be detected28 conjugated to horseradish peroxidase (Bethyl or on clinical signs at the time of a disease investigation. Labs #A100-104 P) were used with 2,2’-azino-bis (3- Sampling was conducted by the Swine Medicine ethylbenzothiazoline-6-sulphonic acid) substrate buffer Education Center. Diagnostics are performed on sera, in citric acid. The reaction was stopped with 5% oral fluid, fecal, and tissue samples. All testing was per- sodium dodecyl sulfate. formed by the ISU VDL.

Monitoring for disease and SPF testing SPF testing. Piglets SF into the STB are evaluated for SPF status at 1 and 2 months of age and must be com- Biosecurity, staff, and PPE. Our facility features dif- pletely negative for all pathogens both times before ferent levels of biosecurity, and PPE guidelines are they can be moved to the LTB. In the LTB they will dependent on the level of biosecurity required. Staff continue to be tested for SPF status at 4, 8, and 12 interacting with animals cannot travel from one level months of age and at six-month intervals thereafter. to a higher biosecurity area without 24 hours’ down Litters produced in the LTB are monitored for SPF time and at least one shower and change of clothing. status at 2 months of age. For SPF testing serum To enter the highest biosecurity region (either the long- blood draws are collected from jugular vein bleeds, term bubble (LTB) or STB), staff must have not been in nasal swabs (BD BBLTM CultureSwabTM Transport contact with outside pigs for at least seven days and a Systems: Liquid Amies, Regular Aluminum Wire, minimum of five showers. Staff remove outer clothing, Fisher Scientific, Part #: B4320129), and fecal samples change into freshly laundered scrubs and socks, then are collected into sterile 50 ml conical tubes. Samples don protective disposable outer shoe covers, zip-up are submitted to the ISU VDL or the Clinical full-body long-sleeved plastic suit (Part #: Pathology Laboratory (see Table 2). 4902,Valumax, PA, USA), surgical mask, hair net, a pair of long cuffed gloves, and a second pair of short cuffed gloves. Although gloves are non-sterile, gloved Results hands are then sprayed with a hydrogen peroxide dis- Large animal biocontainment bubbles infectant and staff immediately enter the high-biosecurity area. For staff moving between animal housing The novel SCID pig ‘‘bubbles’’ create a clean environ- areas, innermost scrubs are protected within full-body ment where SCID pigs can be raised free of pathogen Powell et al. 407

Table 2. Testing for specific pathogen-free (SPF) status from the Veterinary Diagnostic and Clinical Pathology laboratories. Animals are tested for specific porcine pathogens and clinical parameters listed. Testing is conducted from serum, EDTA-treated whole blood, fecal, and/or nasal swab, and diagnostics are performed by the Veterinary Diagnostic or Clinical Pathology Laboratories.a

Test target Type of sample (detection method)

Porcine reproductive and respiratory syndrome Blood serum (ELISA) Porcine parvovirus hemagglutination inhibition Blood serum (ELISA) Mycoplasma hyorhinis Nasal swab (PCR) Mycoplasma hyosynoviae Nasal swab (PCR) Lawsonia intracellularis Fecal, blood serum (ELISA, PCR) Fecal flotation Fecal (sugar float) Swine influenza virus NP Blood serum (ELISA, PCR) Actinobacillus pleuropneumoniae antibody 1-2-9-11 Blood serum (ELISA) Actinobacillus pleuropneumoniae antibody 4-5-7 Blood serum (ELISA) Actinobacillus pleuropneumoniae antibody 3-6-8-15 Blood serum (ELISA) M. hyopneumoniae Fecal, nasal swab (PCR) M. flocculare Fecal, nasal swab (PCR) Porcine epidemic diarrhea virus N gene Fecal, blood serum (ELISA, PCR) Porcine epidemic diarrhea whole virus Fecal, blood serum (ELISA, PCR) Ingezim circovirus Blood serum (ELISA) Rotavirus A, B, C Fecal (PCR) Total blood chemistry panela Blood serum Complete blood counta Blood EDTA

aSamples submitted to the Clinical Pathology Laboratory. EDTA: ethylenediaminetetraacetic acid; NP: nucleoprotein; ELISA: enzyme-linked immunosorbent assay; PCR: polymerase chain reaction. threat (Figure 1(a) and (b)). Air sterility is maintained by sterile rodent box with filter tops for delivery to staff a Biobubble containment structure consisting of a posi- in the bubble. tive-pressure HEPA-filtered airflow system (Figure 1(c)) Upon arrival into the bubble, piglets have cord and UV-treated water system (Figure 1(d)). All person- blood collected and are processed. To provide as safe nel entering the bubbles or interacting with SCID colony an exposure to microbiota as possible, piglets are fed animals wear PPE that minimizes human exposure pasteurized pooled pig colostrum that was collected by as described in the Materials and methods section hand from farrowing animals at the farm from which (Figure 1(e)). See https://vimeo.com/221432512 or con- the carrier sows are originally sourced. To date, pooled tact the Tuggle lab for more information or detailed pasteurized colostrum has been administered to five lit- standard operating procedures. ters and approximately 27 SF piglets. Various pools included samples from 13–45 sows and had an average SF/Cesarean section total IgG concentration of 5.56 (Æ0.13) mg/ml. Since conventional human bottles are inadequate for feeding Protocols for obtaining SPF piglets from non-SPF piglets and difficult to regularly clean in the bubble sows were developed and tested. Piglets obtained by environment, we feed our piglets with a disposable cesarean section are not exposed to vaginal microbiota catheter attached to a 60 ml syringe (Figure 2(a)). We from their sow. To retain a reproductively sound car- tape the tapered end of the tube to our finger and allow rier sow for production of future litters, and produce the piglet to suckle while delivering colostrum with the piglets exposed to maternal microbiota, we developed syringe. This feeding apparatus is disposable, and an SF technique based on prior work.24 Farrowing allows us to carefully monitor and record colostrum sows were moved into a sterile room, washed, and volumes consumed by each individual (Figure 2(b)). monitored for signs of labor. As piglets are delivered, Piglets are fed pasteurized colostrum every three they are caught directly from the vaginal canal in ster- hours until they have consumed 250 ml, at which ile towels by gowned staff and immediately have their point they are fed irradiated milk replacer using the umbilical cords clamped prior to being placed in a same method. Once sufficient colostrum has been 408 Laboratory Animals 52(4)

Figure 2. Snatch-farrowed piglet feeding apparatus. (a) A sterile urinary catheter tube is taped to a gloved finger and attached to a 60 ml tube for feeding. (b) Porcine colostrum or milk replacer is administered as piglets suckle on the finger with tube attached.

Figure 3. ELISA IgG and growth curve data comparing naturally sucking and tube-fed piglets. (a) The box and whisker plot shows serum levels of IgG (mg/ml) from cord blood of SCID (gray) and non-SCID (black) piglets prior to consumption of colostrum compared to SCID and non-SCID samples post-colostrum delivery both from tube-fed and naturally suckling piglets. (NS: non-SCID; S: SCID; NF: naturally farrowed; SF: snatch-farrowed, respectively.) (b) Weight (kg) over time for average of combined SCID and non-SCID piglets that were tube-fed (gray) or naturally suckled (black) is shown. The gray vertical line denotes typical 35-day wean date (from sow or off-milk replacer). Shaded region shows 95% confidence interval. ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; SCID: severe combined immunodeficiency. consumed by all piglets in the litter (within the first 24 cephalic, or ear vein during the first one to four days hours of life), milk replacer is also provided in a separ- of life to determine IgG levels by ELISA (Figure 3(a)). ate feed dish inside the farrowing decks. Piglets typic- Non-SCIDs and SCIDs that were naturally farrowed ally require supplemental tube feeding for the following (NS_NF and S_NF, respectively) had total IgG levels 48–72 hours as they transition to dish feeding. of 13.06 Æ 3.63 mg/ml and 19.05 Æ 4.78 mg/ml, while To confirm we were delivering antibody in pasteur- non-SCID and SCID SF piglets (NS_SF and S_SF, ized colostrum to the bloodstream before gut closure at respectively) had 7.19 Æ 0.51 mg/ml and 11.36 Æ approximately 24 hours of age, we collected serum 4.23 mg/ml (Figure 3(a)). To further compare naturally from cord blood (pre-colostrum) and from the jugular, farrowed versus SF piglets, we examined growth curves Powell et al. 409 for the first 50 days of life from SCID and non-SCID the STB, to the biocontainment transfer cart, and then piglets (Figure 3(b)). Although tube-fed piglets weighed into an internal pen in the LTB (Figure 4(b)). less for the first 30 days, their growth curves were consistent with the trend of naturally suckling piglets and Disease prevalence weight differences are not noticeable after day 35, when piglets were weaned from dam and milk replacer Routine testing of animals at the LCSRC where carrier (shown as vertical line in Figure 3(b)). dams are originally from established that LCSRC pigs carry and are exposed to several common pathogens Animal flow (Table 1). Although the ISU SCID pig is susceptible to virtually all bacterial and viral threats, historically To further reduce the risk of contamination, we estab- the two most common infections documented in our lished a time- and testing-based quarantine system colony are Streptococcus suis and Staphylococcus whereby piglets are tested for SPF status before chan- hyicus, which are common among commercial pig ging location within the facility. To accomplish this, we herds as well as present at the LCSRC (Table 1). utilized two bubbles: the smaller STB that receive SF or Although antibiotic treatment is effective in pigs with cesarean-derived piglets, and the LTB where SPF a normal immune system, we have not observed any females can be raised to sexual maturity. The two bub- SCID piglets to clear the infection, and infected SCID bles are located in adjacent wings of the same building. piglets typically fail to thrive and are euthanized for SF piglets pass through the vaginal canal during far- humane care reasons. The development of the bubble rowing and are thus exposed to maternal microbiota and farrowing system has significantly decreased the and possible contaminants before entering the STB. prevalence and severity of these infections in SF They are tested at 1 month and 2 months of age for SCIDs. While 71% (5/7 litters) of naturally farrowed SPF status (see Materials and methods, Table 2). litters have presented with diagnosed or suspected inci- If either test results in the positive identification of a dences of S. hyicus and/or S. suis, we have identified pathogen in an individual, litter, or pen mate, the pig only two individual cases (2/27 or 8% of piglets) of cannot progress to the LTB. If SPF status is achieved, infection in animals SF into the STB, and zero events female piglets are cleared for transfer to the LTB of infectious disease in our LTB to date. (Figure 4(a)). Additional evidence that high levels of biocontain- To decrease the exposure to potential contaminants ment are necessary for maximum health was provided in the environment outside the positive-pressure by two healthy SCID females that were SF into the bubble, we designed and constructed a battery-oper- STB and maintained SPF status at one and two ated, positive-pressure, HEPA-filtered transfer cart months. These gilts were later moved out of biocontain- that allows us to wheel a pig from an internal pen in ment into non-bubble clean rooms, where they were

Figure 4. Animal transfer through facilities. (a) The figure shows animal flow from farrowing through short- (STB) and long-term bubbles (LTB). Litters are either naturally farrowed where SCID males are BMT rescued and used for subsequent breeding, or snatch farrowed where carrier females are kept SPF clean and moved to the LTB to sexually mature and ultimately be bred to BMT boars to produce naturally farrowed SPF litters in the LTB. (b) HEPA-filtered transfer cart used to move carrier female pigs from the STB into the LTB. SCID: severe combined immunodeficiency; BMT: bone marrow transplantation; SPF: specific pathogen-free; HEPA: high- efficiency particulate air. 410 Laboratory Animals 52(4) cared for following strict clean PPE guidelines, irra- Hinderaker and Eldon Whitaker), the LAR veterinarians diated feed, and the aforementioned monitoring prac- (especially Dr Kathleen Mullin, Dr Amanda Ahrens, and tices. However, within two months after transfer, both Dr Giuseppe Dell’Anna), the Swine Medicine Education animals had positive confirmation of S. hyicus and one Center, the SCID pig team of ISU (especially Jackie Jens, additionally was positive for S. suis. Elizabeth Snella, Adrianne Kaiser-Vry, and Austin Putz), and lastly the Iowa State University Farm staff (especially Gary Kuper). Discussion Declaration of Conflicting Interests The potential contributions of a large-animal SCID research model to the development of regenerative The author(s) declared no potential conflicts of interest with medicine and cancer therapies, as well as improving respect to the research, authorship, and/or publication of this our understanding of the mechanisms of immunity, is article. enormous. The practices described herein focus on the refinement component of the three Rs (replacement, Funding reduction, and refinement) with an emphasis on The author(s) disclosed receipt of the following financial sup- improving the overall management, health, and welfare port for the research, authorship, and/or publication of this of the ISU SCID pig model. article: This work was supported by the National Institutes of The described SCID pig bubbles create a clean envir- Health (grant number 1R24OD019813-1) and the Iowa State onment where SCID pigs can be raised free of pathogen University Vice President for Research. threat. To complete future biomedical studies utilizing SCID pigs, we have developed these facilities for main- References taining an SPF SCID pig colony and concurrently 1. Ozuna AG, Rowland RR, Nietfeld JC, et al. Preliminary developed methods using SF to produce SPF piglets findings of a previously unrecognized porcine primary from non-SPF sows. Our SF protocols and feeding of immunodeficiency disorder. Vet Pathol 2012; 50: pasteurized colostrum successfully delivered IgG to 144–146. neonatal piglets, for which growth follows normal 2. Waide EH, Dekkers JC, Ross JW, et al. Not all SCID pigs are created equally: Two independent mutations in rates seen in naturally suckling piglets. Although Artemis gene found to cause severe combined immuno- some exposure to potential contaminants is possible deficiency (SCID) in pigs. J Immunol 2015; 195: through contact with the vaginal canal of the sow, 3171–3179. our high-sanitation practices are validated by observed 3. Cossu F. Genetics of SCID. Ital J Pediatr 2010; 36: 76. reduced disease incidence and severity. 4. Ewen CL, Cino-Ozuna AG, He H, et al. Analysis of For the long-term SCID pig colony, our goal is to blood leukocytes in a naturally occurring immunodefi- raise females to reproductive age in the LTB, where ciency of pigs shows the defect is localized to B and T they can be artificially inseminated with semen from cells. Vet Immunol Immunopathol 2014; 162: 174–179. our BMT SCID boars to create SPF litters with 50% 5. Powell EJ, Cunnick JE, Knetter SM, et al. NK cells are SCID and 50% non-SCID piglets for further research. intrinsically functional in pigs with severe combined This will decrease labor and the use of resources immunodeficiency (SCID) caused by spontaneous mutations in the Artemis gene. Vet Immunol Immunopathol required by SF and raising piglets in the STB. To 2016; 175: 1–6. date, we have successfully raised four SCID carrier 6. Bosma GC, Custer RP and Bosma MJ. A severe com- gilts to sexual maturity and farrowed a litter within bined immunodeficiency mutation in the mouse. Nature the LTB. The SF practices and unique facilities 1983; 301: 527–530. described herein allow us the flexibility of introducing 7. Perryman LE. Molecular pathology of severe combined new genetics into our herd to control inbreeding, and immunodeficiency in mice, horses, and dogs. Vet Pathol provide us with methods to readily replace animals if 2004; 41: 95–100. necessary or repopulate the LTB in the event of con- 8. Ito R, Takahashi T, Katano I, et al. Current advances in tamination. Furthermore, the development of protocols humanized mouse models. Cell Mol Immunol 2012; 9: that couple SF with biocontainment facilities provides 208–214. valuable information to collaborators who do not have 9. Huang J, Guo X, Fan N, et al. RAG1/2 knockout pigs with severe combined immunodeficiency. J Immunol access to full-scale LTBs, but still require SCID piglets 2014; 193: 1496–1503. for biomedical research. 10. Suzuki S, Iwamoto M, Saito Y, et al. Il2rg gene-targeted severe combined immunodeficiency pigs. Cell Stem Cell Acknowledgments 2012; 10: 753–758. Such a complex project would not be possible without the 11. Lee K, Kwon DN, Ezashi T, et al. Engraftment of human help of a huge team of highly dedicated individuals. We iPS cells and allogeneic porcine cells into pigs with inac- wish to thank the entire LAR staff (especially Dale tivated RAG2 and accompanying severe combined Powell et al. 411

immunodeficiency. Proc Natl Acad Sci U S A 2014; 111: 21. Blanco I, Galina-Pantoja L, Oliveira S, et al. Comparison 7260–7265. between Haemophilus parasuis infection in colostrum- 12. Powell EJ, Cunnick JE and Tuggle CK. SCID pigs: An deprived and sow-reared piglets. Vet Microbiol 2004; emerging large animal model. J Rare Dis Res Treat 2017; 103: 21–27. 2: 1–6. 22. Gomez GG, Phillips O and Goforth RA. Effect of 13. Basel MT, Balivada S, Beck AP, et al. Human xenografts immunoglobulin source on survival, growth, and hema- are not rejected in a naturally occurring immunodeficient tological and immunological variables in pigs. J Anim Sci porcine line: A human tumor model in pigs. Biores Open 1998; 76: 1–7. Access 2012; 1: 63–68. 23. Devillers N, Le Dividich J and Prunier A. Influence of 14. Rajao DS, Loving CL, Waide EH, et al. Pigs with severe colostrum intake on piglet survival and immunity. Animal combined immunodeficiency are impaired in controlling 2011; 5: 1605–1612. influenza A virus infection. J Innate Immun 2017; 9: 24. Huang Y, Haines DM and Harding JC. Snatch-farrowed, 193–202. porcine-colostrum-deprived (SF-pCD) pigs as a model 15. Matsumoto K, Inukai S, Isaka T, et al. Cell counts in for swine infectious disease research. Can J Vet Res peripheral blood and bone marrow of male C.B-17 scid/ 2013; 77: 81–88. scid mice. Lab Anim 1995; 29: 218–222. 25. Chelack BJ, Morley PS and Haines DM. Evaluation of 16. Dietrich HM, Khaschabi D and Albini B. Isolation of methods for dehydration of bovine colostrum for total Enterococcus durans and Pseudomonas aeruginosa in a replacement of normal colostrum in calves. Can Vet J scid mouse colony. Lab Anim 1996; 30: 102–107. 1993; 34: 407–412. 17. Madson DM, Arruda PH, Magstadt DR, et al. 26. Powell EJ, Graham J, Ellinwood NM, et al. T-cell lymph- Characterization of porcine epidemic diarrhea virus iso- oma and leukemia in severe combined immunodeficiency late US/Iowa/18984/2013 infection in 1-day-old cesarean- (SCID) pigs following bone marrow transplantation: derived colostrum-deprived piglets. Vet Pathol 2016; 53: A case report. Front Immunol 2017; 8: 813. 44–52. 27. Quesnel H, Farmer C and Devillers N. Colostrum intake: 18. Lecce JG. Rearing colostrum-free pigs in an automatic Influence on piglet performance and factors of variation. feeding device. J Anim Sci 1969; 28: 27–33. Livest Sci 2012; 146: 105–114. 19. Varley MA, Fowler VR and Maitland A. A rearing 28. Chase C and Polson D. Sampling the herd: When system for colostrum-deprived neonatal piglets. Lab is enough? In: Meeting notes. Proceedings of 31st Anim 1985; 19: 290–296. annual meeting of the American Association of Swine 20. Meyer RC, Bohl EH and Kohler EM. Procurement and Practitioners, Indianapolis, IN, USA, 11–14 March 2000, maintenance of germ-free swine for microbiological p.465. investigations. Appl Microbiol 1964; 12: 295–300.

Re´sume´ Le de´ficit immunitaire combine´ se´ve`re (SCID) est de´fini par l’absence d’un syste`me immunitaire adaptatif. Les mutations entraıˆnant le SCID sont naturellement pre´sentes chez les humains, les souris, les chevaux, les chiens, et rećemment chez les porcs, avec la dećouverte fortuite des porcs atteints de SCID de l’universite´ de l’E´tat d’Iowa. Comme mode`les de recherche, les animaux atteints de SCID tole`rent naturellement la xeńo- transplantation et permettent d’obtenir de prećieux renseignements sur des domaines de recherche tels que la me´decine re´geńe´rative, la the´rapie du cancer, ainsi que les mećanismes de signalisation des cellules immunitaires. Les mode`les biome´dicaux de gros animaux, en particulier les porcs, sont de plus en plus indispensables pour faire progresser l’efficacite´ et l’innocuite´ des nouvelles the´rapies re´geńe´ratrices sur la maladie humaine. Ainsi, il est nećessaire de creér des approches pratiques pour maintenir des mode`les de porcs gravement immunode´prime´s pour la recherche me´dicale exploratoire. Une telle recherche nećessite souvent des lignes geńe´tiques stables pour la re´plication et la survie des animaux sains atteints de SCID pendant des mois apre`s le traitement. Un autre obstacle au de´veloppement des porcs atteints de SCID de l’Universite´ d’Iowa en tant que mode`le biome´dical concernait la mise en place des installations et des protocoles nećessaires a` l’obtention de porcelets SPF issus de l’exploitation porcine classique dans laquelle ils ont e´te´ dećouverts. Une colonie de verrats et truies homozygotes atteints de SCID et porteurs SPF a e´te´ creéé et maintenue en utilisant une reproduction se´lective, des greffes de moelle osseuse, des techniques d’e´levage innovantes et en de´veloppant des installations de bioconfinement.

Abstract Der schwere kombinierte Immundefekt (SCID) wird als fehlende adaptive Immunabwehr definiert. Mutationen, die SCID verursachen, finden sich natu¨rlicherweise bei Menschen, Ma¨usen, Pferden, Hunden und wurden ku¨rzlich auch zufa¨llig bei Schweinen an der Iowa State University entdeckt. SCID-Tiere tolerieren 412 Laboratory Animals 52(4) als Forschungsmodelle von Natur aus Xenotransplantation und liefern wertvolle Erkenntnisse fu¨r Forschungsgebiete wie regenerative Medizin, Krebstherapie sowie Immunzellsignal-Mechanismen. Biomedizinische Großtiermodelle, insbesondere Schweine, werden im Rahmen der angestrebten Verbesserung von Wirksamkeit und Sicherheit neuartiger regenerativer Therapien von Krankheiten des Menschen immer wichtiger. Daher ist es notwendig, praktische Ansa¨tze zur Aufrechterhaltung der Hygiene bei schwer immunsupprimierten Schweinemodellen fu¨r die medizinische Explorationsforschung zu entwick- eln. Fu¨r diese Forschung sind ha¨ufig stabile genetische Linien fu¨r die Replikation und das U¨berleben von gesunden SCID-Tieren Monate nach der Behandlung erforderlich. Eine weitere Hu¨rde bei der Entwicklung des ISU-SCID-Schweins als biomedizinisches Modell war die Schaffung von Einrichtungen und Protokollen, die notwendig sind, um pathogenfreie SPF-Ferkel aus dem konventionellen Schweinezuchtbetrieb zu erhalten, in dem sie entdeckt wurden. Durch selektive Zu¨chtung, Knochenmarktransplantationen, innovative Haltungsverfahren und die Entwicklung von Biocontainment-Einrichtungen wurde eine Kolonie homozygoter SCID-Eber und SPF-Tra¨gersauen geschaffen und aufrechterhalten.

Resumen La Inmunodeficiencia combinada severa (SCID) esta´ definida por la falta de un sistema inmunolo´gico adapt- able. Las mutaciones causantes de SCID se encuentran de forma natural en humanos, roedores, caballos, perros y, recientemente, en cerdos, con el descubrimiento fortuito de cerdos con SCID de la Iowa State University. Al ser modelos de investigacioń, los animales con SCID son tolerantes de forma natural al xenotransplante y ofrecen grandes conocimientos en a´reas de investigacioń como la medicina regenerativa, la terapia para cańcer, adema´s de mecanismos de senãlizaciońdece´lulas inmunes. Los modelos biome´dicos animales de gran tamanõ, en particular los cerdos, son cada vez ma´s esenciales para hacer avanzar la eficacia y la seguridad de terapias regenerativas nuevas sobre enfermedades humanas. Por tanto, existe la necesidad de crear me´todos praćticos para mantener modelos porcinos inmunocomprometidos graves higie´- nicos para la investigaciońme´dica de exploracioń. Esta investigacioń a menudo requiere lıńeas gene´ticas estables para la re´plica y la supervivencia de animales con SCID saludables meses despue´s del tratamiento. Otra dificultad en el desarrollo del cerdo ISU SCID como modelo biome´dico conllevaba el establecimiento de las instalaciones y los protocolos necesarios para obtener cochinillos SPF de la granja convencional de cerdos donde se descubrieron. Una colonia de jabalı´s con SCID homocigo´ticos y reses portadoras de SPF ha sido creada y mantenida mediante crianza selectiva, transplantes de me´dula o´sea, tećnicas de crıá inno- vadoras y el desarrollo de instalaciones de biocontencioń. laboratory an imals Short Report limited Laboratory Animals 2018, Vol. 52(4) 413–417 ! The Author(s) 2018 Brain perfusion fixation in male pigs using Reprints and permissions: sagepub.co.uk/ a safer closed system journalsPermissions.nav DOI: 10.1177/0023677217752747 journals.sagepub.com/home/lan Gracia U. Musigazi1,2, Ste´phanie De Vleeschauwer3, Raf Sciot4, Eric Verbeken4 and Bart Depreitere1,2

Abstract Tissue fixation methods are well established for rodents, but not for large animals. We present a simple technique for in situ brain perfusion fixation in a male porcine model, using cervical vessels for inflow and outflow and achieving a closed system. Thirty-four pigs, aged 4.7 Æ 0.6 months and weighing 60.7 Æ 10.9 kg, were anaesthetised and mechanically ventilated. The ipsilateral common carotid artery and external jugular vein were dissected and constituted the inflow and outflow access, respectively. The brains were perfused and fixed in situ with heparinised saline followed by buffered formaldehyde. Then, specimens (brain, cerebellum and brainstem) were extracted and processed for histology. Fixative fluid leakage was avoided, achieving a closed system. This technique minimises the exposure to toxic chemicals such as formaldehyde and associated hazards (inherent toxicity, eye irritation), thereby increasing operators’ safety. Perfusion was performed with a peristaltic pump for 20–30 minutes at an optimum rate of 0.20 l/min and required only 5 litres of the fixative. The specimens were sufficiently hardened to be extracted. High-quality tissues were available for histology analysis. This technique offers a user-friendly closed system for brain perfusion fixation which can be adapted for other tissues of the head, face and neck.

Keywords brain histology, in situ perfusion fixation, occupational medicine, porcine model

Date received: 26 July 2017; accepted: 5 December 2017

Introduction technique requires massive fluid volumes. Furthermore, it is performed using an open system.7–9 Most fixative The porcine model is one of the best amongst the non- agents are known for their toxicity, and ordinary primate large animals. The pig’s brain is comparable ventilating systems, such as extractor hoods, are not to the human brain with regards to the overall neuro- adapted to the size of large animals. In addition, setting anatomy (gyral pattern, grey/white distribution) and its a vascular access requires a heavy surgical setup and a growth pattern.1,2 Moreover, the brain size and its well-experienced operator.5 biomaterial properties are suitable for computer modelling and simulation.2,3 Fixation perfusion is the gold method for studying 1Experimental Neurosurgery and Neuroanatomy, Neurosciences, the central nervous system and the peripheral nerves. KU Leuven, Belgium 2 The fixative agent penetrates the tissues, ensuring a Department of Neurosurgery, Leuven University Hospitals, Belgium better fixation in addition to maintaining the original 3Laboratory Animal Centre, Biomedical Sciences Group, KU geometry of soft tissues and limiting distortion while Leuven, Belgium 4,5 sampling. Although protocols of perfusion fixation 4Department of Pathology, Leuven University Hospitals, Belgium are well established for rodents, particularly whole- body perfusion fixation via transcardial access,6 fixation Corresponding author: Dr Gracia U. Musigazi, Experimental Neurosurgery and methods for large animals are yet to be established. Neuroanatomy Research Group, KU Leuven, Herestraat 49 – Box Whole-body perfusion fixation in larger animals via 7003, 3000 Leuven, Belgium. transcardial access has several limitations. Firstly, this Emails: [email protected]; [email protected] 414 Laboratory Animals 52(4)

Therefore, we present a simplified technique of in zolazepam (7–10 mg/kg), and then maintained with a situ brain perfusion fixation in a male porcine model, continuous perfusion of intravenous (iv) 20% propofol using the common carotid artery and the external jugu- at 15 ml/min via the auricular veins. Subsequently, the lar vein as inflow and outflow, respectively, and achiev- animals were intubated and mechanically ventilated ing a closed system. (50% oxygen, tidal volume: 600 ml, frequency: 16/min) Thirty-four castrated male domestic pigs, with an to maintain peripheral oxygen saturation above 90% average age of 4.7 0.6 months and average weight and end-tidal carbon dioxide below 40 mm Hg. of 60.7 10.9 kg, were used. These were Landrace, con- Analgesia was achieved with iv buprenorphine ventional and not genetically modified pigs, supplied by (10–20 mg/kg). the Centre for Animal Husbandry, KU Leuven, The pigs were positioned in dorsal recumbency and Belgium. All the animals were delivered by trucks their necks set in extension. Cervical vessels were dis- adapted for large animal transportation at least five sected to set the perfusion connection as illustrated in days before the beginning of the experiments. Figure 1(a)–(e). Two transparent extension tubes were The pigs were housed in individual pens of 2 m2 with used as cannulae (diameter ¼ 3 mm, length ¼ 150 cm, concrete floors and wood shavings as bedding material volume ¼ 1.18 ml). The external jugular vein10 (5 cm in an open system (no special microbiological protect- below the fork at the junction of the linguofacial vein ive measures). All cages had a hanging toy. The follow- and maxillary vein) was used for the outflow as ing parameters were checked and documented daily. illustrated in Figure 1(b). The liquids were drained by Firstly, 14-hour light/10-hour dark cycle from 7:00 gravity into 5 litre plastic containers set on the floor am to 9:00 pm. Secondly, temperature was maintained (Figure 1(e)). The common carotid artery,10 running at 22 2C. Thirdly, humidity ranged between 45% deeper along the trachea with the internal jugular vein and 70% but could vary with weather conditions, espe- and the vagus nerve branch (Figure 1(a)), was used as cially in winter. All animals had free access to tap water inflow and was connected to a peristaltic pump through an automated drinking system. The pigs (Figure 1(d)). The perfusion tubes were inserted up to received Optivo Pro 9041Õ (AVEVE, Leuven, a length of 4–5 cm inside the vessels and restricted by Belgium) pellets twice daily. surgical strands (2-0 MersileneÕ Ethicon, Somerville, Our study was approved by the Ethical Committee NJ, USA) as shown in Figure 1(c). for Animal Experimentation, KU Leuven (P160/2014, Following this, iv heparin (10,000 IU) was adminis- since 1 September 2014) and all the animal experiments tered and the animals were euthanised with an excess were carried out at the Laboratory Animal Centre, KU of barbiturates. Death was confirmed after the oxygen Leuven, Belgium. saturation and end-tidal carbon dioxide curves General anaesthesia was induced with intramuscular flattened on the monitoring device. The brain was 20% lidocaine (0.05–0.3 mg/kg) and tiletamine and immediately perfused with a rinsing solution of 3

Figure 1. (a)–(e) Closed system of perfusion developed in-house – fixation for tissues of the head (brain and face) using cervical neck vessels by means of a unilateral perfusion. (a) The common carotid artery (arrow down) with the internal jugular vein and the vagus nerve. (b) The external jugular vein (arrow up). (c) Perfusion connections: common carotid artery as inflow and external jugular vein as outflow. (d) Inflow sustained by a pump. (e) Liquid drainage into a plastic container. PFA: phosphate-buffered formaldehyde; NaCl: sodium chloride. Musigazi et al. 415

Figure 2. (a)–(h) High-quality histology material produced after in situ perfusion fixation. (a) Frontal, parietal, temporal and occipital bone pieces removed, exposing a fixed brain covered by dura mater. (b) Specimen removed from the skull. (c) and (d) Pig brain tissues fixed and processed for paraffin embedding. (e) and (f) Fronto-parietal sample of porcine dura mater. (g) and (h) Frontal cerebral cortex. Magnification: (e) and (g): 12.5; (f) and (h): 400.

litres of heparinised saline and then fixed in situ with 5 We have presented a simplified and user-friendly litres of 4% solution of phosphate-buffered formalde- closed system in a porcine model for large-animal hyde (neutral pH) at 0.20 l/min (Figure 1(b)). Next, the brain perfusion fixation. To the best of our knowledge, inflow and outflow tubes were clamped to let formal- this is the first time that successful in situ brain dehyde penetrate the tissues. The pig head was then cut perfusion fixation has been reported in a porcine off. All the muscles and connective tissue were removed model using the unilateral common carotid artery and and the skull was carefully sawed allowing the specimen external jugular vein. So far, perfusion fixation in large (brain, cerebellum and brainstem) to be delicately animals has been performed with a bilateral vascular removed along with the meningeal coverings (dura access and massive fixative volumes.7–9 and pia mater). Finally, the brain tissues were processed Following a skin incision,10 the targeted vessels for histology analysis. can be quickly found, dissected and cannulated, The pigs were perfused successfully in situ and allowing for a fast procedure. Moreover, this closed good quality brain fixation was obtained in a reprodu- system increases the safety of the operators handling cible way. toxic fixative solutions. Lastly, this technique has the Leakage of fixative fluid was avoided by properly potential to be used for brain fixation of other large connecting the perfusion tubes to the vessels of the animals. neck, thereby achieving a nearly closed system Mathematical and computer models offer a valu- (Figure 1(a)–(e)). Because the system was powered by able alternative to animal experiments (in silico a peristaltic pump, a relatively rapid perfusion at an approach). Once a mathematical model is validated, optimum rate of 0.20 l/min could be performed. computer simulation not only allows significant This technique minimises exposure to chemical sub- reduction in the number of animals used in experi- stances such as formaldehyde and health hazards mentation but also speeds up what can be performed (inherent toxicity, eye irritation) associated with it, in the lab. However, the accuracy of such models thereby increasing operators’ safety. depends on the data used as input.11 The ultimate A good perfusion was indicated by homogeneous goal of our work is to provide detailed histology brain tissue firmness and a change of colour from data that can be implemented into such computer orange to yellow.4 Indeed, the brain tissues were har- models. dened well enough after the in situ perfusion fixation to allow the required manipulation during brain extraction (Figure 2(a)–(d)). Thus, high-quality brain tissues Acknowledgements were available for histology analysis as illustrated in We would like to thank the Laboratory Animal Centre, Figure 2(e)–(h). KU Leuven, Leuven, Belgium, as the animals were housed 416 Laboratory Animals 52(4) and taken care of by their staff during the experiments. References Next, we highly appreciate the support provided by the 1. Duhaime AC, Margulies SS, Durham SR, et al. Departments of Pathology and Forensic Medicine, Maturation-dependent response of the piglet brain to Leuven University Hospitals. Especially, we acknowledge scaled cortical impact. J Neurosurg 2000; 93: 455–462. Dr Wim Develter for sharing his precious expertise in 2. Manley GT, Rosenthal G, Lam M, et al. Controlled cor- handling brain autopsy. We are also grateful to Sijn tical impact in swine: Pathophysiology and biomechanics. Massart and all his colleagues at the Centre for Animal J Neurotrauma 2006; 23: 128–139. Husbandry in Lovenjoel for their collaboration in breed- 3. Prange MT and Margulies SS. Regional, directional and ing, choosing and delivering the animals. Lastly, Marc age-dependant properties of the brain undergoing large Verbeek, from the Medical Technical Services at Leuven deformation. J Biomech Eng 2002; 124: 244–252. University Hospitals, has also been of great help in 4. Williams TH and Jew JY. An improved method for per- adapting some of the devices we needed to process the fusion fixation of neural tissues for electron microscopy. pig brain histology. Tissue Cell 1975; 7: 407–418. 5. Fix AS and Garman RH. Practical aspects of neuropath- Declaration of Conflicting Interests ology: A technical guide for working with the nervous The author(s) declared no potential conflicts of interest with system. Exp Toxicol Pathol 2000; 28: 122–131. respect to the research, authorship, and/or publication of this 6. Gage GJ, Kipke DR and Shain W. Whole animal perfu- article. sion fixation for rodents. J Vis Exp 2012; 65: 3564. doi: 10.3791/3564. Funding 7. Simmons MM, Blamire IW and Austin AR. Simple method for the perfusion-fixation of adult bovine brain. The author(s) disclosed receipt of the following financial sup- Res Vet Sci 1996; 60: 247–250. port for the research, authorship, and/or publication of this 8. Finnie JW, Blumbergs PC, Manavis J, et al. Evaluation article: This technique was developed for a multidisciplinary of brain damage resulting from penetrating and non- project supported by the Fonds voor Wetenschappelijk penetrating captive bolt stunning using lambs. Aust Vet Onderzoek – Vlaanderen (Research Foundation-Flanders) J 2000; 78: 775–778. grant number: G.0C6713.N) and by the KU Leuven (grant 9. Manger PR, Pillay P, Maseko BC, et al. Acquisition of number: OT/12/071). brains from the African elephant (Loxodonta africana): Perfusion-fixation and dissection. J Neurosci Methods Contributions 2009; 179: 16–21. GUM: conception and design, acquisition, analysis and 10. Popesko P. Atlas of topographical anatomy of the domestic interpretation of data, drafting and reviewing the article, animals. Vol 1. 5th ed. Philadelphia, PA: WB Saunders final approval. SDV: conception and design, reviewing the Company, 1986, pp.88–99. article, final approval. RS: analysis and interpretation of 11. Famaey N, Ying Cui Z, Umuhire Musigazi G, et al. data, reviewing the article, final approval. EV: conception Structural and mechanical characterisation of bridging and design, final approval; and BD: conception, final veins: A review. J Mech Behav Biomed Mater 2015; 41: approval. 222–240.

Re´sume´ Les me´thodes histologiques de fixation des tissus sont bien e´tablies pour les rongeurs, mais c’est n’est le cas pour les gros animaux. Nous pre´sentons une technique simple pour la fixation du cerveau par perfusion in situ, dans un mode`le porcin de sexe masculin, en utilisant les vaisseaux cervicaux comme voies d’acce`set produisant ainsi un syste`me ferme´. Trente-quatre porcs, aˆge´s de 4,7 0,6 mois et pesant 60,7 10,9 kg, ont e´te´ anesthe´sies et mećaniquement ventile´s. L’arte`re carotide commune et la veine jugulaire externe homo- late´rale, ont e´te´ disse´queés et choisies comme voie d’acce`s et d’e´vacuation, respectivement. Les cerveaux ont e´te´ perfuse´s et fixe´s in situ avec une solution saline he´parineé, suivie par une solution de formalde´hyde tamponneé. Puis, les tissus (cerveau, cervelet, et tronc ce´re´bral) ont e´te´ extraits et pre´pare´s pour l’analyse histologique. La fuite de solution de fixation a e´te´ e´viteé graˆce a` ce syste`me ferme´. De ce fait, cette technique, minimisant l’exposition aux produits chimiques toxiques, tel que le formalde´hyde, et a` leurs dangers associe´s (toxicite´ intrinse`que, irritation oculaire), augmente la sećurite´ des utilisateurs. La perfusion a e´te´ effectueé graˆce a` une pompe pe´ristaltique pendant 20 a` 30 minutes, a` une vitesse optimale de 0,20 l/min, et avec seulement 5 litres de solution de fixation. Les tissues e´taient suffisamment fixe´s pour en permettre l’extraction. Nous avons obtenues des tissus de haute qualite´ pour l’analyse histologique. Cette technique de´voile un syste`me ferme´, utilise´ pour la fixation du cerveau par perfusion in situ, pouvant eˆtre adapteé pour d’autres tissus de la teˆte, du visage et du cou. Musigazi et al. 417

Abstract Es existieren ga¨ngige Methoden zur Gewebefixierung bei Nagetieren, jedoch nicht bei Großtieren. Wir stellen eine einfache Technik fu¨r die in situ Hirnperfusionsfixierung bei einem ma¨nnlichen Schweinemodell vor, bei der Halsgefa¨ße fu¨r den Zu- und Abfluss verwendet werden und ein geschlossenes System erzielt wird. Vierunddreißig Schweine im Alter von 4.7 0.6 Monaten und einem Gewicht von 60.7 10.9 kg wurden beta¨ubt und mechanisch belu¨ftet. Ipsilaterale Karotis und a¨ußere Halsschlagader wurden seziert und bildeten jeweils den Zu- bzw. Abflusszugang. Die Gehirne wurden perfundiert und in situ mit heparinisierter Kochsalzlo¨sung fixiert, gefolgt von gepuffertem Formaldehyd. Anschließend wurden Proben (Gehirn, Kleinhirn und Hirnstamm) extrahiert und fu¨r die Histologie aufbereitet. Fixierflu¨ssigkeitsleckagen wurden vermieden, wodurch ein geschlossenes System erreicht wurde. Diese Technik minimiert die Exposition gegenu¨ber tox- ischen Chemikalien wie Formaldehyd und den damit verbundenen Gefahren (inha¨rente Toxizita¨t, Augenreizung) und erho¨ht so die Sicherheit des Personals. Die Perfusion wurde mit einer Peristaltikpumpe fu¨r 20–30 Minuten bei einer optimalen Rate von 0.20 l/min durchgefu¨hrt, wobei nur 5 Liter des Fixiermittels beno¨tigt wurden. Die Proben waren ausreichend geha¨rtet, um extrahiert werden zu ko¨nnen. Fu¨r die histolo- gische Analyse stand hochwertiges Gewebe zur Verfu¨gung. Diese Technik bietet ein benutzerfreundliches geschlossenes System zur Hirnperfusionsfixierung, das an anderes Gewebe des Kopfes, des Gesichts und des Halses angepasst werden kann.

Resumen Existen me´todos de fijacioń de tejido consolidados para roedores pero no para animales ma´s grandes. Presentamos una tećnica simple para una fijacioń de perfusioń cerebral in situ en un modelo porcino macho, utilizando vasos cervicales para entrada y salida y para conseguir un sistema cerrado. Treinta y cuatro cerdos de edades comprendidas entre 4.7 0.6 meses y un peso de 60.7 10.9 kg fueron anestesiados y ventilados mecańicamente. La arteria caro´tida comuń ipsilateral y la vena yugular externa fueron diseccio- nadas y constituyeron el acceso de entrada y salida, respectivamente. Los cerebros fueron perfundidos y fijados in situ con suero heparinizado seguido de formaldehı´do tamponado. Ma´s tarde, las muestras (cerebro, cerebelo y tronco encefa´lico) fueron extraı´das y procesadas para la histologıá. Se evito´ una fuga de fluido fijador, consiguiendo de este modo un sistema cerrado. Esta tećnica minimiza la exposicioń a productos quı´micos to´xicos como formaldehı´do y otros peligros relacionados (toxicidad inherente, irritacioń ocular), aumentando de este modo la seguridad del operador. La perfusioń se realizo´ con una bomba perista´ltica durante 20–30 minutos a un ritmo o´ptimo de 0.20 l/min, y se requirieron tan solo 5 litros de fijador. Las muestras estaban lo suficientemente duras como para ser extraı´das. Habıá disponible tejidos de alta calidad para un ana´lisis de histologıá. Esta tećnica ofrece un sistema cerrado sencillo para la fijacioń mediante perfusioń cerebral, que tambień puede adaptarse para otros tejidos de la cabeza, cara y cuello. laboratory an imals Case Report limited Laboratory Animals 2018, Vol. 52(4) 418–423 ! The Author(s) 2018 Malignant lymphoma with middle ear Reprints and permissions: sagepub.co.uk/ involvement in a Sprague-Dawley rat journalsPermissions.nav DOI: 10.1177/0023677218764768 journals.sagepub.com/home/lan Jean-Francois Lafond and Annick Landry

Abstract Sprague-Dawley rats are amongst the most widely used animals in biomedical research and malignant lymphoma has long been known to be a frequent neoplasm in these animals. A 9-month-old male control Sprague-Dawley rat from a toxicity study showed gelatinous material in the cranial cavity and dark, thickened cerebral meninges at necropsy. At microscopic evaluation of the temporal bone, neoplastic lymphocytes were seen invading several structures of the middle ear. The neoplastic cells appeared to extend from the marrow of the temporal bone, covered the dorsal part of the tympanic cavity wall, and surrounded and infiltrated the base of the tensor tympani muscle as well as the chorda tympani branch of the facial nerve. The lymphoma was generalized; neoplastic lymphocytes were also noted in numerous other tissues. Literature regarding neoplasms of the middle and inner ear in animals is scarce and, to our knowledge, this is the first report of a lymphoma involving the middle ear of a rat.

Keywords Rodents, organisms and models, pathology, neoplasia, toxicology

Date received: 31 October 2017; accepted: 20 February 2018

Introduction The animal was group-housed with two other control group males in a polycarbonate cage containing appro- Sprague-Dawley rats are amongst the most widely used priate bedding, a hiding tube and chewing object, and animals in biomedical research and malignant lymph- equipped with an automatic watering valve. The room oma has long been known to be a frequent neoplasm in temperature was kept between 19 and 25C, the humid- aged, and even middle-aged, individuals of this strain. ity was between 30 and 70%, and the light cycle was The incidence of malignant lymphoma in aged 12 hours of light and 12 hours of darkness. The animal Sprague-Dawley rats was reported to be close to 2% was fed PMI Nutrition International Certified Rodent in males and up to slightly over 4% in females.1,2 In Chow No. 5CR4 ad libitum. All animal procedures male Sprague-Dawley rats up to 50 weeks old, this neo- were conducted with guidance from the USA plasm was identified as the most common tumor.3 National Research Council and the Canadian Council Malignant lymphomas in rats are well known to gener- on Animal Care, and under approval from the alize to most organs but, to our knowledge, invasion of Institutional Animal Care and Use Committee of the rat middle ear by this neoplasm has never been Charles River Laboratories. reported. The day before scheduled necropsy, the animal weighed 786 g compared to a group mean of 890.4 g Case presentation and showed a 4% body weight loss during the previous A 9-month-old male Sprague-Dawley rat (Charles River Canada Inc., St-Constant, Quebec, Canada) Charles River Laboratories, Canada from a 26-week oral gavage toxicity study was assigned Corresponding author: to a group-housed control group and was administered Jean-Francois Lafond, Charles River Laboratories, 22022 a non-pharmacologically active, non-toxic reference Transcanadienne, Senneville, Que´bec, Canada, H9X 3R3 item (citrate buffer in ultra pure water) daily. Email: [email protected] Lafond and Landry 419 week, after a constant weight gain during the rest of the muscle, as well as the chorda tympani branch of the study. facial nerve (Figure 3). The neoplastic lymphocytes The animal was euthanized by exsanguination from were large, with scant eosinophilic to amphophilic cyto- the abdominal aorta after isoflurane anesthesia as plasm and large nuclei, which often contained several planned at the end of the study, with no clinical signs conspicuous nucleoli (Figure 4). Mitotic figures and other than oily fur consistent with decreased grooming. necrosis of individual neoplastic cells were frequently A complete necropsy examination was performed and observed. The malleus was the only ossicle present in included evaluation of the carcass and musculoskeletal the sections examined and was not infiltrated by the system, all external surfaces and orifices, the cranial neoplastic cells. The tympanic membrane and external cavity and external surfaces of the brain and the thor- ear canal appeared normal. The portions of the inner acic, abdominal and pelvic cavities with their associated ear that were present in the sections examined, includ- organs and tissues. Macroscopically, dark and gelatin- ing part of the vestibule and cochlea, did not contain ous material was observed in the cranial cavity over the any neoplastic cells. The lymphoma was generalized temporal bone and the cerebral meninges were thick- and neoplastic lymphocytes were also noted in the adre- ened and dark. Additionally, the spleen was enlarged nal glands, bone marrow, brain, femur, kidneys, larynx, and there were dark foci on the mandibular lymph liver, lymph nodes, meninges, nasal cavity, pituitary nodes. A standard and exhaustive list of tissues, along gland, prostate, spinal cord, spleen, sternum and thy- with the grossly abnormal meninges including the gel- roid glands. In the spleen and mandibular lymph nodes, atinous material described above and underlying tem- the lymphoma correlated microscopically with the poral bone, were sampled at necropsy, fixed in 10% macroscopic lesions described above (i.e. enlarged buffered formalin, sectioned, and stained with hema- spleen and dark foci in the lymph nodes). toxylin and eosin for histopathological evaluation. In order to elucidate the origin of the gelatinous material, Discussion microscopic evaluation of the temporal bone was performed, and neoplastic lymphocytes were seen invading The most common primary neoplasms of the middle several structures of the middle ear (Figure 1). These and inner ear in humans are endolymphatic-sac neoplastic cells appeared to extend from the marrow of tumors, squamous carcinomas, adenocarcinomas, the temporal bone and formed a sheet several cells thick rhabdomyosarcomas, multiple myelomas, plasmacyto- that covered the dorsal part of the tympanic cavity wall mas and lymphomas,3,4 and the middle ear is known to (Figure 2). Medially, neoplastic lymphocytes also sur- be a primary location of some carcinomas of the exter- rounded and infiltrated the base of the tensor tympani nal auditory canal.5 Middle and inner ear involvement

Figure 1. Outer, middle and inner ear with multifocal invasion of neoplastic lymphocytes (*). Hematoxylin and eosin stain. Left inset corresponds to Figure 3. Right inset corresponds to Figure 2. Scale bar, 500 mm. C: cochlea; E: external ear canal; M: malleus; TC: tympanic cavity; TM: tympanic membrane; V: vestibule. 420 Laboratory Animals 52(4) in human lymphoma has been well described and the icterus and a palpably enlarged spleen.11 At necropsy, neoplastic lymphocytes are thought to reach these areas typical findings include splenomegaly, hepatomegaly either following inner ear hemorrhage or by direct inva- and lymphadenomegaly,12 which correlate microscop- sion of the solid structures of the middle ear, body ically with infiltration by neoplastic lymphocytes. labyrinth and inner ear.6–10 Because of the complicated tridimensional anatomy Clinically, rats with malignant lymphoma will show of the middle and inner ear, and the difficult dissection, mainly non-specific signs such as depression, pallor, trimming and processing inherent to those anatomic

Figure 2. Extension of neoplastic lymphocytes (*) from the bone marrow into the tympanic cavity lumen. Hematoxylin and eosin stain. Inset corresponds to Figure 4. Scale bar, 250 mm. BM: bone marrow; M: malleus; TC: tympanic cavity lumen.

Figure 3. Neoplastic lymphocytes (*) surrounding and infiltrating the base of the tensor tympani muscle and the chorda tympani branch of the facial nerve. Hematoxylin and eosin stain. Scale bar, 250 mm. CT: chorda tympani branch of the facial nerve; TC: tympanic cavity; TT: tensor tympani muscle; V: vestibule. Lafond and Landry 421

Figure 4. Higher magnification showing large neoplastic lymphocytes (*) with scant cytoplasm and large nuclei infiltrating the tympanic cavity. Hematoxylin and eosin stain. Scale bar, 100 mm. TB: temporal bone; TC: tympanic cavity. features, these structures are often overlooked at nec- Acknowledgement ropsy and are seldom part of histopathological exam- The authors would like to thank Hamid Boubekeur for his ination. Thus, literature regarding neoplasms of the invaluable help with the images. middle and inner ear in animals is scarce and, to our knowledge, this is the first report of a naturally- occuring lymphoma involving the middle ear of a rat. Declaration of conflicting interests However, in Wistar rats, an experimental model of The author(s) declared no potential conflicts of interest with infiltration of the inner ear scala tympani through the respect to the research, authorship, and/or publication of this cochlear aqueduct following inoculation of neoplastic article. lymphocytes into the cisterna magna has been 13 described. Carcinomas of Zymbal’s gland, a small Funding auditory sebaceous gland seen in mice as well,14 is a 15 The author(s) received no financial support for the research, common ear tumor in rats but originates from the authorship and/or publication of this article. external ear and, to our knowledge, is not known to infiltrate the middle ear. Most animal middle ear neoplasms reported appear References to have been seen in dogs and were most frequently 1. Chandra M, Riley M and Johnson D. Spontaneous neo- extensions of primary growths that originated in the plasms in aged Sprague-Dawley rats. Arch Toxicol 1992; external ear canal, including adnexal tumors and 66: 496–502. adenocarcinomas of the ceruminous glands.16 Of 2. Nakazawa M, Tawaratani T, Uchimoto H, et al. note, a single case of lymphoma occupying the tym- Spontaneous neoplastic lesions in aged Sprague-Dawley panic cavity was reported in a cat.17 rats. Exp Anim 2001; 50: 99–103. 3. Son W and Gopinath C. Early occurrence of spontaneous In conclusion, even though there was no such occur- tumors in CD-1 mice and Sprague-Dawley rats. Toxicol rence in the present case, lymphoma with inner ear Pathol 2004; 32: 371–374. involvement should be added to the list of possible 4. Devaney K, Boschman C, Willard S, et al. Tumours of the diagnoses in middle-aged and older rats showing external ear and temporal bone. Lancet Oncol 2005; 6: signs of vestibular disorder, along with the frequent 411–420. pituitary gland neoplasms, in particular pars distalis 5. Kollert M, Draf W, Minovi A, et al. Carcinoma of the adenomas. external auditory canal and middle ear: therapeutic 422 Laboratory Animals 52(4)

strategy and follow up. Laryngorhinootologie 2004; 83: 12. Percy D and Barthold S. Pathology of laboratory rodents 818–823. and rabbits. Ames: Blackwell Publishing, 2007, p.170. 6. Hustert B, Stoll W and August C. Tumors of the tem- 13. Kano M. An experimental study of tumor cell infiltration poral bone: a primary non-Hodgkin lymphoma of the into temporal bones. Nippon Jibiinkoka Gakkai kaiho internal auditory canal and the cerebellopontine angle. 1998; 101: 884–894. Laryngorhinootologie 1997; 76: 625–628. 14. Seely J and Boorman G. Mammary gland and specialized 7. Okura S and Kaga K. Temporal bone pathology of leu- sebaceous glands (Zymbal, preputial, clitoral, anal). kemia and malignant lymphoma with middle ear effu- In: Maronpot R, et al (eds) Pathology of the mouse. sion. Auris Nasus Larynx 1994; 21: 1–7. Reference and atlas. Vienna: Cache River Press, 1999, 8. Scott S, Burgess R, Weber P, et al. Non-Hodgkin’s pp.613–635. lymphoma of the middle ear cleft. Otolaryngol Head 15. Rudman D, Cardiff R, Chouinard L, et al. Proliferative Neck Surg 1997; 6: S203–S205. and nonproliferative lesions of the rat and mouse mam- 9. Merchant S and McKenna M. Neoplastic growth. mary, Zymbal’s, preputial and clitoral glands. Toxicol In: Merchant S and Nadol J (eds) Schuknecht’s pathology Pathol 2012; 40: 7s–39s. of the ear. Shelton: People’s Medical Publishing House, 16. Little C, Pearson G and Lane J. Neoplasia involving the 2010, p.532. middle ear cavity of dogs. Vet Rec 1989; 124: 54–57. 10. Michaels L and Hellquist H. Neoplasms of the inner ear. 17. Delorimier L, Alexander S and Fan T. T-cell lymphoma Ear, nose and throat histopathology. London: Springer- of the tympanic bulla in a feline leukemia virus-negative Verlag, 2001, p.132. cat. Can Vet J 2003; 44: 987–989. 11. Boorman G and Everitt J. Neoplastic disease. In: Suckow M, Weisbroth S and Franklin C (eds) The laboratory rat. Burlington: Elsevier Academic Press, 2005, p. 498.

Re´sume´ Les rats Sprague-Dawley sont parmi les animaux les plus utilise´s en recherche biome´dicale et on sait depuis longtemps que le lymphome malin est un neóplasme fre´quent chez ces animaux. L’autopsie d’un rat Sprague-Dawley controˆle de 9 mois, issu d’une e´tude de toxicite´,are´ve´le´ une matie`re ge´latineuse dans la cavite´ craˆnienne ainsi que des meńinges ce´re´brales sombres et e´paissies. Lors de l’e´valuation microscopique de l’os temporal, des lymphocytes neóplasiques envahissant plusieurs structures de l’oreille moyenne ont e´te´ observe´s. Les cellules neóplasiques infiltraient la moelle de l’os temporal, couvraient la partie dorsale de la paroi de la cavite´ tympanique et entouraient en l’infiltrant la base du muscle tenseur du tympan ainsi que la branche chorda tympani du nerf facial. Le lymphome e´tait geńe´ralise´; les lymphocytes neóplasiques e´taient pre´sents dans de nombreux autres tissus. La litte´rature sur les neóplasmes de l’oreille moyenne et interne chez l’animal est rare et il s’agit a` notre connaissance du premier rapport portant sur un lymphome impli- quant l’oreille moyenne d’un rat.

Abstract Sprague-Dawley-Ratten geho¨ren zu den am haüfigsten in der biomedizinischen Forschung verwendeten Tieren und das maligne Lymphom ist seit langem als haüfiges Neoplasma bei diesen Tieren bekannt. Eine 9 Monate alte ma¨nnliche Sprague-Dawley-Kontrollratte aus einer Toxizita¨tsstudie wies bei der Sektion gel- atino¨ses Material in der Scha¨delho¨hle und dunkle, verdickte Hirnhaüte auf. Bei der mikroskopischen Beurteilung des Schla¨fenbeins wurden in mehrere Strukturen des Mittelohrs eingedrungene neoplastische Lymphozyten beobachtet. Die neoplastischen Zellen schienen sich aus dem Mark des Schla¨fenbeins zu erstrecken, bedeckten den dorsalen Teil der Trommelfellwand und umgaben und infiltrierten die Basis des Musculus Tensor tympani sowie den Chorda tympani-Zweig des Gesichtsnervs. Das Lymphom wurde general- isiert; neoplastische Lymphozyten wurden auch in zahlreichen anderen Geweben beobachtet. Literatur u¨ber Neoplasmen des Mittel- und Innenohrs bei Tieren ist rar und nach unserem Kenntnisstand ist dies der erste Bericht u¨ber ein Lymphom unter Beteiligung des Mittelohrs bei einer Ratte.

Resumen Las ratas Sprague-Dawley son entre los animales ma´s utilizados en la investigacioń biome´dica y hace anõs que se sabe que el linfoma maligno es un neoplasma frecuente en estos animales. Una rata Sprague-Dawley Lafond and Landry 423 de control macho y de 9 meses utilizada en un estudio de toxicidad presento´ un material gelatinoso en la cavidad craneal y meninges cerebrales oscuras y gruesas en la necropsia. Durante la evaluacioń microsco´- pica del hueso temporal, se observaron que los linfocitos neopla´sticos invadıán varias estructuras del oı´do medio. Las ce´lulas neopla´sticas parecıán extenderse de la me´dula del hueso temporal, cubrir la parte dorsal de la pared de la cavidad timpańica y rodear e infiltrarse en la base del mu´sculo timpańico tensor adema´sde la cuerda del tı´mpano del nervio facial. El linfoma fue generalizado; tambień se observaron linfocitos neopla´sticos en muchos otros tejidos. No hay muchos documentos sobre neoplasmas del oı´do interior o medio y seguń nuestro parecer este es el primer registro de un linfoma que cubre el oı´do medio de una rata. Sign up for FREE updates about the latest research! journals.sagepub.com/action/registration

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FELASA is the Federation of European Laboratory . American Association for Laboratory Animal Animal Science Associations and a LAL Association Science–FELASA working group liaisons, including Member. It is a recognised and respected representative a report on harm–benefit analysis of animal studies; of Laboratory Animal Science (LAS) in Europe and . a worldwide recognised course accreditation scheme beyond. However, FELASA may mean different for education and training to promote best practice things to different people, organisations and countries. and endorse the 3Rs (Replacement, Reduction and When asked about FELASA’s notable achievements, Refinement); its National Association members cited among other . recommendations for education and training (origin- things its collaborative work, including support ally on FELASA Categories A, B, C and D and later for new associations, to promote high LAS standards to be superseded by recommendations on the EU and contribution to raise LAS education Functions); standards. FELASA Membership allows its Member . Severity Classification Workshops, supported and Associations to be in contact with a broad community partially funded by FELASA; of specialist scientists in the field of LAS and facilitates . since 1981, the organisation of triennial congresses the exchanging of ideas and experiences. that offer continuing education opportunities for FELASA promotes best practice in LAS and animal delegates; and welfare and sets widely recognised standards for scien- . developments of links across Europe and beyond tists involved in animal research thanks to: with associations and governmental bodies.

. working groups on key topics; All of FELASA’s activities remain true to its mission to . recommendations on health monitoring; promote best practice in LAS and animal welfare and . guidelines and reports on animal welfare and ethical this will continue as FELASA embarks on its next review; 40 years in serving the LAS community.

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www.ipsltd.biz laboratory an imals News limited Laboratory Animals 2018, Vol. 52(4) 429 ! The Author(s) 2018 Reprints and permissions: Workshop sobre Clasificacio´n de Severidad sagepub.co.uk/ journalsPermissions.nav en Madrid DOI: 10.1177/0023677218788799 journals.sagepub.com/home/lan

Elena Hevia

El pasado 5 de Abril tuvo lugar en Madrid, en el Centro based on the following topic ‘Severity Classification Nacional de Biotecnologıá (CSIC) el taller de FELASA and Reporting under EU Directive 2010/63 / EU’. titulado ‘‘Severity Classification and Reporting under The workshop was organized by Dr. Beleń Pintado EU Directive 2010/63/EU’’. Este taller lo organizo´la and lectures were provided by Anne-Dominique Dra. Beleń Pintado y fue impartido por Anne- Degryse and Jose´Ma Orellana in collaboration with Dominique Degryse y Jose´Ma Orellana con la cola- Alberto Pastor. There were 40 attendees from different boracioń de Alberto Pastor, y a e´l asistieron 40 per- disciplines: animal welfare managers, researcher sonas entre las que se encontraban responsables de groups, ethic committee members, and representatives animalarios, investigadores, miembros de comite´sde from the authorities in this matter, at both regional and e´tica y representantes de las autoridades en la materia national levels. tanto a nivel regional como nacional. The workshop was highly interactive, allowing the El worshop fue muy interactivo y en e´l se estudiaron various groups to make severity evaluations on differ- varios supuestos praćticos sobre los que los grupos ent practical cases, and bringing these together, before hicieron valoraciones de la severidad, para despue´s finally listening to FELASA’s evaluation. hacer una puesta en comuńyoı´r la valoracioń dada These activities are always found interesting and are por FELASA. very well received by professionals in the industry Este tipo de actividades siempre son interesantes y aiming to unify (severity) assessment criteria. bien acogidas por los profesionales interesados en uni- These kinds of workshops will be repeated in differ- ficar los criterios de valoracioń en la medida de lo ent locations in our country in order to reach as many posible. people as possible who may be interested in this matter. Este taller se repetiraén distintas ubicaciones de We will be providing further information on how they nuestra geografıá para que llegue al mayor nu´mero develop after they take place. posible de las personas interesadas. Iremos dando informaciońdeco´mo se ha desarrollado en las distintas sedes.

Workshop on Severity Classification in Madrid On April 5, 2018, a FELASA workshop was held at Madrid’s National Centre of Biotechnology (CSIC)

SECAL (Sociedad Espan˜ola para las Ciencias del Animal de Laboratorio), Spain

Corresponding author: Elena Hevia, SECAL, Spain. Email: [email protected]

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The query for quality and translation of experimental animal studies 15 - 16 October 2018, Barcelona Spain

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"0;;+1&"4*,,.&& *<2=>>+(.38?+4.3;@,17> +:0435,->+(.38?+4.3;? (0;;+1?(4*,,.>& laboratory an imals News limited Laboratory Animals 2018, Vol. 52(4) 436–437 ! The Author(s) 2018 Reprints and permissions: ECLAM/ESLAV Pre-Conference Workshop: sagepub.co.uk/ journalsPermissions.nav DOI: 10.1177/0023677218788794 Publishing Your Science: Toolkit and journals.sagepub.com/home/lan Guidelines

ESLAV-ECLAM AALAC-SECAL Conference 2018 Registration: E90; limited to 12 participants Date: Sunday, 14 October 2018 Contact: Janet Rodgers, ECLAM Secretariat Place: Barcelona, Spain; deﬁnitive location will be com- ([email protected]) municated to participants in due course

Agenda Time Title Topic Speaker

08:00–08:30 Arrival and registration check 08:30–10:30 Scientific Writing Toolkit The building blocks of expository writing Simon Bartlett 10:30–11:00 Coffee break 11:00–12:30 Scientific Writing Toolkit The shape of a research article: Simon Bartlett what belongs where; textual strategies for writing the introduction, results, methods and figure legends. 12:30–13:30 Lunch 13:30–15:30 Scientific Writing Toolkit The shape of a research article: continued Simon Bartlett 15:30–16:00 Coffee break 16:00–17:30 ARRIVE guidelines Reporting guidelines Merel Ritskes-Hoitinga

Correspondence: Janet Rodgers, Box 314, 266 Banbury Road, Oxford, OX2 7DL, UK. Email: [email protected] News 437

Scientific writing toolkit B. The shape of a research article: what belongs Simon Bartlett1 where; textual strategies in writing the introduction, results, methods and figure legends. Writing for The sessions and exercises are organised to aid partici- cohesion: Applying the ideas explored to the analysis pants in producing publications that meet the standards and revision of example texts from research articles. for professional journals. The course deals with ways to create cohesion in expository prose in English, with a . Stylistic requirements for different research article specific focus on the biomedical sciences. The goal is to sections. IMRD structure and variants. equip participants with a set of ‘mental tools’ that . Research article introductions: the CARS (creating a together provide a clearly structured way of thinking research space) model about how a text is constructed, from the internal com- . Methods, Results and Figure Legend exercise: recog- ponents of the sentence through to paragraphs and nising different content and stylistic requirements of the distinct sections of a research article. The sessions these complementary texts. and exercises are organised such that participants dis- . Overview of Abstracts and Discussion sections (if cover for themselves how to gain control over their time allows) writing by paying close attention to their experience . Reference and online resources as readers. The aim is for the sessions to be as interactive as possible, with participants encouraged to ask questions Reporting guidelines: prepare in time and to provide their own insights and ideas. Merel Ritskes-Hoitinga2. When writing scientific art- A. The building blocks of expository writing. The icles, it is very important to write down all the details, exercises look at a series of overlapping and comple- in order for readers to know exactly what you have mentary themes related to how we choose which types been doing and to be able to replicate studies in case of content belong in the different locations of a text. needed. Also, when performing systematic reviews, it is These exercises are organized broadly as follows: essential to know the quality of the details of the included primary studies in order to make high quality . Events, action and agency: How do we identify and evidence-based evaluations. There are several reporting convey the essential message of a sentence? guidelines for animal studies available (Gold Standard . Character: Choosing how to begin a sentence; why it Publication Checklist, ARRIVE, HARRP, etc.), which matters and what criteria we need to apply. will be presented. Participants will get a scientific pub- . Interruptions, separations and clutter: Some lication from a high-profile journal and evaluate this common problems that arise in the internal structure article against a checklist with publication require- of sentences and how to avoid them. ments. Possible reasons for the current low implemen- . Beyond the sentence: tation of reporting guidelines will be discussed and (i) Beginnings: How to ensure a fluid link at the possible solutions. In order to write good publications, start of a sentence from the preceding material. it is necessary to prepare well in advance before starting The difference between subject and topic. and designing animal studies. The PREPARE guide- Different kinds of linker structure and how to lines have been developed for achieving exactly that. use them. (ii) Endings: What do we remember about a Notes sentence? 1. Simon Bartlett is Scientific Editor at Centro Nacional de (iii) Points of emphasis, and how they are signalled Investigaciones Cardiovasculares, Madrid, Spain. with punctuation and specific clause patterns. 2. http://www.syrcle.nl/; http://www.ritskes-hoitinga.eu/ Providing leadership in laboratory animal science and welfare in support of ethical and effective Laboratory Animal animal research Science Association The professional society for those with an interest in... Animal use • Care and Welfare Education and training • 3Rs • Ethics Animal research regulation and policy JOIN TODAY! Benefits of being a member: • Membership of a highly regarded and influential scientific association • Access to support for career development at all levels • The opportunity to serve on high level Sections and Task Forces charged with delivering scientific guidance and policy proposals • Preferential registration for the Annual Conference and other events • Access to Association bursaries, travel awards and CPD opportunities • Regular newsletter: ‘LASA Forum’

www.lasa.co.uk/join_us [email protected] Explore the Laboratory Animals Handbooks

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Laboratory Animals

The international journal of laboratory animal science and welfare, Laboratory Animals publishes peer-reviewed original papers and reviews on all aspects of the use of animals in biomedical research. The journal promotes improvements in the welfare or well-being of the animals used, particularly focusing on research that reduces the number of animals used or which replaces animal models with in vitro alternatives.

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journals.sagepub.com/home/lan ...... 440 Calendar of events

Meetings of interest to laboratory animals scientists and technicians: references to Laboratory Animals are for further details. Items for inclusion should be sent to Notes and Comments Editor, LAL, PO Box 373, Eye, Suffolk, IP22 9BS, UK. Email to [email protected]. The deadlines for inclusion of material are: February issue, 10 November; April issue, 10 January; June issue, 10 March; August issue, 10 May; October issue, 10 July; December issue, 10 September.

2018

23–24 August 2nd Pan-American Conference for Alternative Methods, Rio de Janeiro, Brazil. For further information visit http://caat. jhsph.edu/programs/workshops/PanAmerican2018/index.html 27–29 August Biobarriers, 2018, Saarbru¨cken, Germany. For further information visit http://biobarriers.hips-wordpress.helmholtz-hzi. de/ 2–5 September 54th Congress of the European Societies of Toxicology (EUROTOX 2018), Brussels, Belgium. For further information visit http://www.eurotox-congress.com/2018/ 12–14 September GV-SOLAS Annual Conference, Munich, Germany. For further information visit For further information visit http://www. gv-solas2018.de/# 19 September The LASA/UFAW 3Rs Section Meeting - Applying the 3Rs to the breeding of animals for research purposes, Stevenage, UK. For further information email [email protected] 23–26 September EUSAAT 21st European Congress on Alternatives to Animal Testing, Linz, Austria. For further information visit http:// www.eusaat-congress.eu/ 30 September–3 October Safety Pharmacology Society 2018 Annual Meeting, Washington, DC. For further information visit https://www. safetypharmacology.org/AM2018/ 15–16 October ESLAV-ECLAM-AAALAC International-SECAL Conference, Barcelona, Spain. For further information visithttp://www. barcelonacongress2018.com/ 15–16 October Modeling the Mammalian Microbiota Host Superorganism, Current Tools and Challenges, Paris, France. For further information visit http://www.mam-2018.org/home 15–18 October 20th International Congress on In Vitro Toxicology 2018, Berlin, Germany. For further information visit http://www. estiv2018.com/ 28 October–1 November AALAS National meeting, Baltimore, Maryland. For further information visit https://www.aalas.org/national-meeting/ general-information/future-meetings 30 October RSPCA/UFAW Rodent and Rabbit Welfare Meeting, London, UK. For further information visit [email protected] 4–7 November American College of Toxicology Annual Meeting (ACT 2018), Palm Beach, Florida. For further information visit https:// www.actox.org/am/am2018/splashpage.asp 20–21 November SGV Meeting 2018, Lausanne, Switzerland. For further information visit https://naturalsciences.ch/organisations/sgv/ meetings 27–29 November LASA Annual Conference, Birmingham, UK. For further information contact [email protected]

2019

9–12 April Institute of Animal Technology (IAT) Congress, North West England, UK. For further information visit http://www.iat. org.uk/congress 10–13 June 14th FELASA Congress, Prague, Czech Republic. For further information visit http://www.felasa2019.eu/

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