3 Minute 3Rs

It's the December episode of 3-Minute 3Rs, brought to you by , the NC3Rs (www.nc3rs.org.uk), the North American 3Rs Collaborative (www.na3rsc.org) and Lab Animal (www.nature.com/laban)The papers behind the pod:1. Optimizing the design of population-based patient-derived tumor xenograft studies to better predict clinical response https://bit.ly/2GwGJ7N2. An In Vitro Model of Hematotoxicity https://bit.ly/2Ezgaw33. Found In Translation https://go.nature.com/2UVJ0g6[NC3Rs] Every cancerous tumor is different and treating two tumors with the same drug may result in two very different outcomes, which contributes to the high failure rate during cancer drug discovery. One experiment type gaining interest to model this tumor heterogeneity is screening a population of patient-derived tumor xenograft models, or PDX models. However, there is little guidance available for these experiments. This risks poor experimental design that could result in a study being under or overpowered. Both these scenarios waste animals. A new freely available interactive tool from a team at AstraZeneca, published in Disease Models & Mechanisms, enables scientists to design robust and reproducible population PDX studies. The tool developed by Floc’h et al explores the design of population PDX studies and how this can impact the risk of both false-negative and false-positive results. Each experiment for a specific drug or cancer type is unique so the tool cannot provide exact recommendations, but what it can do is support you in selecting the optimal design for your experiment whilst ensuring the 3Rs are also considered. Details for how to access the tool can be found via the link in the description.[NA3RsC] Mature blood cells and bone marrow are common targets of drug toxicity. In addition to leading to significant complications for patients receiving antineoplastic agents for cancer treatment, these hematotoxic effects can be limiting, preventing administration of the doses needed for therapeutic efficacy. Screening for potential toxicity to the hematopoietic system in animals and in human cell-based in vitro approaches is critical in early drug development. The CFU assay has been validated as the traditional screen for hematotoxicity, but has limitations. A publication in Current Protocols in Toxicology by Mahalingaiah and colleagues describes the next-generation, high-throughput assay as a refined approach. A liquid culture, lineage-specific, in vitro cell differentiation model offers multiple advantages over the traditional assay, making it a versatile and useful tool to screen compounds. In addition to decreasing the number of animals required for hematoxicity determination, this human, cell-based in vitro assay is a useful alternative to in vivo studies for investigative work in understanding mechanisms of lineage-specific hematotoxicity at different stages of differentiation.[LA] So you’ve made a mouse model of disease, applied a treatment, and measured gene expression. Now what? You might think the gene with the greatest changes in expression is going to be the most important for the disease. But there’s a pretty big evolutionary gap between mouse and man. From just your experiment, do you really know how relevant that gene will be? To better bridge the species gap, researchers from Technion have developed a new statistical model that takes decades of previously recorded gene expression data in mouse and human into account. They call it Found in Translation, or FIT for short. For new mouse data, FIT calculates a per-gene effect size and predicts which gene in your mouse might be most relevant to humans. It can potentially rescue genes that might not otherwise make the cut for further studies and de-prioritize those important to a mouse but perhaps less so to a person. No new mice experiments required. You can find all the details about FIT in Nature Methods, and you can try it out yourself with your own gene expression data at mouse2man.org.

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It's the November episode of 3-Minute 3Rs, brought to you by Lab Animal (www.nature.com/laban), the NC3Rs (www.nc3rs.org.uk) & the North American 3Rs Collaborative (www.na3rsc.org)The papers behind the pod:1. Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci. https://www.nature.com/articles/s41588-018-0223-82. Effects of Cage Enrichment on Behavior, Welfare and Outcome Variability in Female Mice https://www.frontiersin.org/articles/10.3389/fnbeh.2018.00232/full3. Effects of Music Enrichment on Individually Housed Male New Zealand White Rabbits. https://doi.org/10.30802/AALAS-JAALAS-17-000153Transcript:[NC3Rs] When the complete mouse genome sequence was first published 16 years ago, it was a breakthrough that sped up many scientific discoveries and improved our understanding of genetics. But, given the large collection and variety of mouse strains available, it is very important to recognise and understand the differences between them. This new study by Lilue et al, published in Nature Genetics, strives to do just that. Collaborators from 21 institutes produced draft genome reference sequences, from scratch, for 16 of the most widely used inbred mouse strains. They found extensive strain-specific variation, particularly in regions impacting immune response, sensory function, sexual reproduction and behaviour. This is a powerful resource for mouse genetics that will help scientists appreciate, for instance, why mouse strains respond differently when studying their immune system or behaviour. I personally really liked this work because even though the 3Rs might not have been the authors’ primary motive, the outcomes will have important 3Rs implications, giving researchers cause to more carefully consider which strain or model they choose to answer their experimental question, and avoiding the wastage of animals. The genomes can be freely accessed and analysed online. To find out more, visit the link in the description. [LA] ...Different countries have different husbandry standards, and questions linger among researchers about whether that environmental variability can lead to experimental variability too. For the past five years, Hanno Wurbel at the U. of Bern... has been systematically assessing lab mouse husbandry standards. Earlier in the year, he and his colleagues reported on cage sizes. In their latest work, they’ve turned their attention to cage enrichment. Working with female inbred C57BL6 and outbred SWISS mice, they tested a variety of outcomes across four enrichment levels, from cages with only bedding to highly enriched, semi-naturalistic ones with lots of vertical height and structures for the mice to interact with. Measures of welfare increased with greater enrichment, with no evidence of increased variation in experimental results. Read the full report in the journal Frontiers in Behavioral Neuroscience, and then give some more thought to giving your mice some more structure.[NA3RC] The beneficial effect of music has been shown across a variety species and is often used as a form of enrichment for rabbits in the laboratory setting, but is it really music to their ears? Species-specific music is based on the theory that music with a tempo that matches an animal’s internal circadian rhythms, brain waves, heart rate and respiration can promote relaxation. In a study by Jessica Peveler and Debra Hickman in the November issue of JAALAS, the effect of commercially available music for rabbits was evaluated. Older male rabbits were exposed to music enrichment for 6 months. A decrease in the stress hormone, cortisol, was seen when music was provided, with cortisol returning to baseline levels after the music was removed. These findings suggest that commercially available music can decrease stress and be used as a refinement to improve well-being of rabbits used in biomedical research.

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Welcome to the October episode of 3-Minute 3Rs, brought to you by Lab Animal (www.nature.com/laban, the NC3Rs (www.nc3rs.org.uk) & the North American 3Rs Collaborative (www.na3rsc.org)Here are the papers behind the pod:1. Generation of Tumor-Reactive T Cells by Co-culture of Peripheral Blood Lymphocytes and Tumor Organoids. https://www.cell.com/cell/fulltext/S0092-8674(18)30903-62. Mymou: A low-cost, wireless touchscreen system for automated training of nonhuman primates. https://link.springer.com/article/10.3758%2Fs13428-018-1109-53. Practical rat tickling: Determining an efficient and effective dosage of heterospecific play. https://www.appliedanimalbehaviour.com/article/S0168-1591(18)30255-7/fulltextTranscript:[LA] Organoids are three-dimensional tissue cultures that resemble more complicated structures. They're promising alternatives to animal models for studying diseases and for testing potential treatments. But tissues grown in a dish lack an immune system. That's a critical component for predicting how something will function in a living organism. To add some immunity to their organoids, Emile Voest from the Netherlands Cancer Institute and his colleagues recently developed a new co-culturing platform. In their latest work, published in the journal Cell in September, they culture epithelial organoids, derived from human cancer patients, along with blood from those same patients. The co-culturing platform allowed the researchers to study how T-cells from the patients’ immune systems interacted with the organoids, attacking cancerous cells while ignoring healthy ones. It’s a personal approach for understanding immunotherapy, no animals required.[NA3RsC] We all strive to provide laboratory animals with positive welfare, but it’s difficult to actually know how animals feel. But with rats, we have a unique window into their emotional lives through their vocalizations. Rats make positive ultrasonic vocalizations when they play and when they’re tickled. What is tickling? It mimics rat rough and tumble play, which rats will eagerly seek out. But how much time does this technique take? A recent publication by LaFollette et al, gives us that answer. Researchers tickled rats for a frequency of 1, 3, or 5 days and for a duration of 15, 30, or 60 seconds. They found that 3-days of tickling was the most efficient and effective. These rats produced more positive vocalizations before and during tickling, played more, and were less inactive in their home cage before tickling. However the duration of tickling didn’t matter. This suggests that 3 short sessions of tickling can go a long way to make rats happier to interact with humans and to improve rat welfare.[NC3Rs] Many neuroscientific studies involve training nonhuman primates to perform cognitive tasks. But traditional training methods raise some key concerns. These include the need to separate animals from their home environment, causing them stress that affects both their welfare and their performance. Enter Mymou, a refined training system developed by James Butler and Steve Kennerley at University College London and described in a recent Behavior Research Methods paper. Mymou, named after the Greek word for "monkey" is a wireless touchscreen system that is installed in the home environment. It runs continuously, so monkeys can train whenever they want in familiar surroundings. Built in facial recognition allows different tasks to be assigned to different individuals while real-time data analysis means tasks can be adapted as training progresses. The set-up uses commercially available components and costs under £400, or around 500 USD. If that wasn't enough, Mymou is also fully open source, making it even easier to try out in any lab. Following the link in the description to find out more.

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Welcome to the September episode of 3-Minute 3Rs, brought to you by Lab Animal (www.nature.com/laban), the NC3Rs (www.nc3rs.org.uk) & the North American 3Rs Collaborative (www.na3rsc.org)Here are the papers behind the pod:1. Assessing animal affect: an automated and self-initiated judgment bias task based on natural investigative behaviour. https://doi.org/10.1038/s41598-018-30571-x2. Comparison of serial blood collection by facial vein and retrobulbar methods in C57BL/6 mice. https://doi.org/10.30802/AALAS-JAALAS-17-0001343. Can grimace scales estimate the pain status in horses and mice? A statistical approach to identify a classifier. https://doi.org/10.1371/journal.pone.0200339Transcript:[LA]...Your valence, that is, whether you’re feeling positive or negative, can influence your judgment. There’s evidence that animals are no different, and how an animal responds to an ambiguous cue could a way to gauge their affective state and refine their welfare. But testing for judgment biases can be time consuming for the people involved. In August, researchers at the University of Bristol published an automated judgment bias test involving a natural rat behavior in the journal Scientific Reports. They developed a set up to train rats without human intervention to associate one audio tone with a food reward and another with an unpleasant puff of air. The rats have to nose poke into a food trough to start a trial, and either leave their nose in place or withdraw it according to what they hear. How they respond to ambiguous tones should reflect their valence. The cues still need a bit more work, but rats are at least up to the challenge of learning such an automated judgment bias task that could potential be used for future welfare research. [NA3RsC] Serial blood collection in mice is often necessary for sentinel testing and for tracking experimental processes. In July, Jennifer Frohlich and her colleagues published a study in JAALAS that could help refine the process of recurring blood draws in mice. The study, Comparison of Serial Blood Collection by Facial Vein and Retrobulbar Methods, performed single and serial blood draws for both methods. The single facial vein group experienced the least clinical signs after blood collection and the least histological lesions. The single and serial retrobulbar groups both had a similar amount of harderian adenitis and ocular trauma on histology. Meanwhile, the serial facial vein group had a 33% mortality rate, the most clinical signs post-collection which included seizures, head tilt, and convulsions; and 2 mice had histologic brain lesions consistent with trauma. The researchers concluded that the facial vein method is less desirable for serial blood collection in mice compared with the retrobulbar method.[NC3Rs] Effective, accurate pain assessment is key to safeguarding the welfare of any animal. And one such approach growing in use among animal researchers are grimace scales. Using this methods, individuals observe specific expressions associated with pain in a particular species and score them collectively for their intensity. The higher the score, the greater the pain the animal is perceived to be in. Of course, it is vital to validate that any method of pain assessment is effective and reliable. In her paper published this August in PLoS One, Dr. Emanuela Dalla Costa, from the University of Milan, has done just this. In her study, Dr. Dalla Costa collected over 1000 grimace scores from individual observations of horses and mice, pre- and post-surgery. Their data was subjected to exhaustive statistical analysis to determine how the level of pain the animal was experiencing, and the variation in the different observers used, influenced the overall pain score the animals received. The analysis identified both mouse and horse grimace scales to a high level of consistency across each of the individual users, demonstrating these scales to be a reliable method of assessing pain...

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You're listening to the August edition of 3 Minute 3Rs, brought to you this month by the NC3Rs (www.nc3rs.org.uk), the North American 3Rs Collaborative (www.na3rsc.org), and Lab Animal (www.nature.com/laban) This month, we'll be talking about non-primate alternatives to study polio, aging & frailty indexes, and training rats with out water restriction.The papers behind the pod:* Replacement of primary monkey kidney cells by L20B cell line in the test for effective inactivation of inactivated poliovirus vaccine:https://www.sciencedirect.com/science/article/pii/S0166093417306006* Senolytics improve physical function and increase lifespan in old age: http://www.nature.com/articles/s41591-018-0092-9* A Comparison of Two Mouse Frailty Assessment Tools: https://academic.oup.com/biomedgerontology/article/72/7/904/3854927* Training Rats Using Water Rewards Without Water Restriction: https://www.frontiersin.org/articles/10.3389/fnbeh.2018.00084/full

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You're listening to the July edition of 3 Minute 3Rs, brought to you this month by the North American 3Rs Collaborative (www.na3rsc.org), Lab Animal (www.nature.com/laban) and the NC3Rs (www.nc3rs.org.uk). This month, we'll be talking about refining mouse anesthesia, replacing mice with zebrafish for studying human hematopoetic stem cells, and validating genome-edited mouse lines.The papers behind the pod:* Effects of Ketamine Compared with Urethane Anesthesia on Vestibular Sensory Evoked Potentials and Systemic Physiology in Mice: http://www.ingentaconnect.com/content/aalas/jaalas/2018/00000057/00000003/art00007;jsessionid=2je0i010idu59.x-ic-live-03* A method for transplantation of human HSCs into zebrafish, to replace humanised murine transplantation models :https://f1000research.com/articles/7-594/v1* Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-018-0530-7

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You're listening to the June edition of 3 Minute 3Rs, brought to you this month by Lab Animal (www.nature.com/laban), the NC3Rs (www.nc3rs.org.uk) and the North American 3Rs Collaborative (www.na3rsc.org). We're highlighting Drosophila as a microbiome model, organotypic brain culture slices for Alzheimer's research, and anesthesia for African clawed frogs.The papers behind the pod:* The Drosophila model for microbiome research: https://www.nature.com/articles/s41684-018-0065-0* Preparation of organotypic brain slice cultures for the study of Alzheimer’s disease: https://f1000research.com/articles/7-592/v1* Comparison of Etomidate, Benzocaine, and MS222 Anesthesia with and without Subsequent Flunixin Meglumine Analgesia in African Clawed Frogs (Xenopus laevis): http://www.ingentaconnect.com/contentone/aalas/jaalas/2018/00000057/00000002/art00013

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Welcome to 3-minute 3Rs for May 2018! in this podcast, we're discussing enrichment, anesthesia for cephalopods, and reproducibility across labs.The papers behind the pod:Laboratory mouse housing conditions can be improved using common environmental enrichment without compromising data: http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2005019In Vivo Recording of Neural and Behavioral Correlates of Anesthesia Induction, Reversal, and Euthanasia in Cephalopod Molluscs: https://www.frontiersin.org/articles/10.3389/fphys.2018.00109/fullReproducibility of preclinical animal research improves with heterogeneity of study samples: http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2003693And be sure to visit www.nature.com/laban and www.nc3rs.org.uk for more information about your hosts today.

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Welcome to 3-minute 3Rs! In this podcast, we're discussing 'dirty' mice, automating pain assessments, and refining endpoints with telemetry data.The papers behind the pod:News Feature: What happens when lab animals go wild: http://www.pnas.org/content/115/13/3196A deep neural network to assess spontaneous pain from mouse facial expressions: http://journals.sagepub.com/doi/10.1177/1744806918763658Using Telemetry Data to Refine Endpoints for New Zealand White Rabbits Challenged with Bacillus anthracis https://www.ncbi.nlm.nih.gov/pubmed/29256375

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There's over 1300 species of bat in the world, and Bat 1K (http://bat1k.ucd.ie/) wants to sequence them all. Hear from director Emma Teeling from University College Dublin about the consortium's goals and strategies, and why understanding bats better is so important.