3 Minute 3Rs

The October 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).Papers1. Modelling human hepato-biliary-pancreatic organogenesis from the foregut–midgut boundaryhttps://www.nature.com/articles/s41586-019-1598-02. Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms https://www.nature.com/articles/s41598-019-50920-83. Analysis of vertebrate vision in a 384-well imaging system. Thorn RJ. Sci Rep. https://www.ncbi.nlm.nih.gov/pubmed/31562366Transcript[LA] Organoids are three dimensional structures derived from human cells. Unlike traditional cell cultures that are grown flat on petri dishes, multicellular organoids can offer researchers a little extra physiological context. But, an organoid of one organ still exists in isolation. A new paper published last month in the journal Nature starts to put some organoids together.The team, led by Takanori Takebe at Cincinnati Children’s Hospital, wanted to study organagenesis, the process by which organs form. They took human skin cells, reverted them to pluripotent stem cells, and meticulously guided those to become small balls called spheroids that mimicked foregut and midgut. The team then put the two kinds of spheroids together in a special medium and let them grow. The structure that ultimately formed recapitulated functions of the liver, bile ducts, and pancreas. Such an integrated system is another step forward for organoid research. [NA3RsC] The worm, C. elegans, is an increasingly popular model for drug discovery and screening. For example, adverse effects of compounds can easily be detected by looking for changes in how fast the C. elegans grow, how big they get, and once they are larvae - how they move. Atakan and colleagues present a platform for automating the process of phenotyping single C elegans from the embryo to the adult. The platform overcomes many of the challenges of prior systems. It automates the painstaking process of distributing embryos into individual chambers and can expose the worms to up to 8 different chemical conditions in a single experiment. The phenotypes, including growth and movement, are measured automatically through analysis of 10 second videos captured every four hours across the worms’ life cycle. To validate the platform, C. elegans were exposed to increasing concentrations of tetramisole and the known negative effects on growth and motility were demonstrated. The continued progress toward automating C. elegans studies with platforms, such as this one, will allow scientists to discover new opportunities for using the worm in early drug discovery. [NC3Rs] Automated behavioural monitoring can reveal subtle changes in an animal’s vision before they can be detected physically. While these tests can be applied to rodents in the lab, each animal must be individually trained to use the system and only one animal can be monitored per cage, making it impractical for high throughput studies. In search of an alternative approach, Robert Thorn and colleagues at Brown University turned to zebrafish larvae at five days post-fertilisation. In their recent Scientific Reports paper, the team describe imaging 384 larvae at once in four 96-well plates, monitoring their response to moving visual stimuli. They found that diazepam significantly reduced their response, while flumazenil, a diazepam antidote, reversed this effect, illustrating that the larvae can be used to detect clinically relevant changes in the visual system, With three percent of the world’s population affected by visual impairment, this new tool could speed up the screening and development of treatments while also replacing mammalian models.

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The September 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)Papers behind the pod:1. http://jpet.aspetjournals.org/content/371/1/15.long2. https://www.sciencedirect.com/science/article/pii/S001216061830006X3. https://www.nature.com/articles/s41467-019-11259-w[NC3Rs] Accurate measurements of drug and metabolite concentrations in the blood are vital to estimate exposure to the target in humans and animals during drug development. Zebrafish larvae are increasingly used for pharmacological research, but measurements of blood drug concentrations in these small animals have been technically challenging.A team at Leiden University have developed a method for nanoscale blood sampling from the posterior cardinal vein of zebrafish larvae at five days post fertilisation. A median volume of 1.12 nL of blood can be collected from each embryo and samples pooled to form a single replicate, which can be analysed by Liquid chromatography–mass spectrometry. While drug and metabolites could be successfully measured in the pooled samples, the authors suggest improvements to the sensitivity of the technique, which could reduce the number of embryos needed for each replicate. In addition, using microfluidic embryo handling techniques, blood sampling could be further automated and yield improved while reducing the amount of drug required. Further development of this microsampling technique has the potential to increase the use of the zebrafish embryo model to define drug pharmacokinetic properties. [NA3RsC] Josephine Morris and her colleagues at Bristol University used transgenic lines of zebrafish to study mechanisms of collagen formation and repair. This work was published in the journal Developmental Biology, Vol 441. They crossed transgenic zebrafish lines which integrated green fluorescent protein, expressed in the epidermis and mCherry collagen which is specifically expressed in the basal epidermis which allowed them to understand the dynamic nature of collagen 1 fibril deposition. The authors used Transmission Electron microscopy to demonstrate the intricate pattern integration of the fluorescent proteins in embryonic development. In other studies, a wound was created with a 30 g needle on the flank of 4 day old larvae and collagen repair was documented pictorially. By using the GFP collagen and mCherry collagen lines together, they were able to exploit the unique live imaging in larval fish to probe the process of collagen deposition and wound remodeling specifically in the basal epidermis and deeper layers. It is hoped that these transgenic lines will enable live imaging of collagen deposition and remodeling in various other organs and diseases.[LA] And finally, say hello to LipoGlo, a new reporter system for keeping track of lipoproteins in vivo. These proteins ferry fats throughout the body – you may be familiar with HDLs and LDLs, the latter of which can contribute to cardiovascular disease in people. A particular particle, Apolipoprotein B-containing lipoprotein, is particularly problematic and while it has been studied in mammals, such animals don’t lend themselves to the large numbers needed for high-throughput drug discovery work. Enter the larval zebrafish. In order to visualize APoB-lipoproteins across the whole translucent organism, the research team took advantage of a glowing enzyme called NanoLuc, which they attached to APoB in the larvae. This bioluminescent reporter is highly sensitive and quite bright, allowing the team to follow the distribution and concentrations of the lipoprotein as it traversed the vascular system of the tiny little zebrafish. Additional details about the new tool can be found in the journal Nature Communications.

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The August episode of 3-Minute 3Rs.Papers:1. https://advances.sciencemag.org/content/5/7/eaaw40992. https://www.ncbi.nlm.nih.gov/pubmed/310260403. https://www.ncbi.nlm.nih.gov/pubmed/31343844[LA] Nociception is the sense that tells animals they are encountering a potentially damaging stimulus. In people, we call it pain. Most pain research is done with rodents, but nociception is pretty well conserved beyond mammals. That includes invertebrates, such as the fruit fly. Some work has been done in larval drosophila, but larvae exist in a transient state; any quote unquote pain is therefore transient too. To study longer lasting changes in nociception, University of Sydney researcher Greg Neely recent;y described his lab’s efforts to develop an adult fly model. They took advantage of the fly’s natural aversion to surface temperatures above 42 degrees Celsius, and show that after peripheral injury-in this case, a leg amputation-the flys thermal sensitivity changes in a chronic manner. Looking deeper, they saw evidence of a complex mechanism known as central disinhibition at work, along with a role for a conserved gene called Twist in mediating that chain of events.You can read more about this potential fly replacement for nociception studies in the journal Scientific Advances.[NC3Rs] Before starting an experiment, a researcher should always have its end in mind. What is the hypothesis? The experimental objectives? And crucially for animal experiments, what signs indicate the experiment should be stopped to prevent unnecessary suffering? It’s a challenging task though, identifying appropriate humane endpoints, as these should be specific to the animal and model but aren’t always described in detail in the literature. Computers, however, are notoriously good at identifying patterns so researchers at the University of Berlin have investigated whether machine learning could be used to define humane endpoints in mouse models of stroke and sepsis. Data was collated from previous studies, focussing on commonly applied humane endpoints such as loss of body weight, changes in body temperature and the use of a sickness severity score. Using these measures, the machine learning based approach was able to predict whether an animal had a high risk of death, with up to 93% accuracy in the mouse model for stroke and 96% accuracy for sepsis. Notably, machine learning could make these predictions one to two days before a humane endpoint would be reached. And the faster we can identify an animal suffering or in pain, the faster we can intervene.[NA3RsC] A paper in Journal of BioPhotonics sheds some light on the value of fibered confocal fluorescence microscopy. Fibered confocal fluorescence microscopy is a confocal microscope equipped with a fiber-optic bundle which provides light to organs and tissues, enabling collection of real-time images of biologic processes in live animals. With conventional confocal microscopes, cell imaging is limited due to the difficulty with accessing deep tissues and organs. However, advancements in fiber-optic imaging technologies enables improved visualization of tissues and organs, including blood vessels, vasculature and nerves, and mucosal surfaces of the colon. In addition to the many scientific advantages, fibered confocal fluorescence microscopy also enables 3Rs impact. Each animal can be used as its own control with more information obtained from fewer animals, reducing the total number of animals required and inter-animal variability while increasing statistical validity. Also, in vivo imaging is non-invasive providing a valuable refinement to enabling identification of tissues under pathological conditions and responses to drug treatments. Finally, imaging of human organs and tissues could ultimately replace the use of animals, leading to improved clinical relevance making this a valuable translational tool expected to have increased impact in drug research and development.

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The July episode of 3-Minute 3Rs from the North American 3Rs Collaborative (www.na3rsc.org), the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)Papers; 1. https://advances.sciencemag.org/content/5/5/eaav65282. https://www.nature.com/articles/s41598-019-44705-23. https://www.nature.com/articles/s41598-019-45464-w[NC3Rs] Colorectal cancer is particularly hard to treat, and only 11% of patients whose tumours have metastasised survive five years after diagnosis. Mouse models that incorporate patient tissue are widely used in drug discovery, but they’re both expensive and time consuming, and often don’t fully represent human disease. A team at the Mount Sinai Hospital in New York, led by Erdem Bangi, developed a screening platform using Drosophila to test potential therapies for a patient whose cancer had proved resistant to treatment. The team identified nine genes that were driving the progression of the disease, so they modified orthologs of these genes in the flies. They were able to use the flies to screen a raft of drug combinations, discovering that the most effective treatment combined trametinib, a chemotherapy drug, and zoledronate, usually used to treat bone diseases. When the patient was treated with this novel combination, their tumours shrank, then remained stable for a total of 11 months. This platform could reduce the number of mice used to find effective cancer treatments, not to mention getting them to patients quicker and cheaper. Personalised medicine is often said to be the next big thing in healthcare, but this new approach might just live up to the buzz.[NA3RsC] Too much standardization of experimental design may be unintentionally contributing to the reproducibility crisis in animal-based research. Today, rigorously standardized behavioral studies may be so well controlled that statistically significant findings may actually be irrelevant and not reproducible across laboratories. Bodden and colleagues at the University of Münster, in Germany, explore whether systematically varying study conditions can improve reproducibility. They specifically investigated the impact of the time of day the testing occurred on the outcomes of five different behavioral tasks across several replicates. They found that behavioral task results differed significantly depending on the time of day animals were tested, morning, noon, or afternoon. Follow-up simulations using the data generated suggest that systematically introducing two testing times, both morning and afternoon, into the same experimental design can significantly improve reproducibility of results within the same lab and, more importantly, is predicted to lead to better reproducibility across laboratories. This study describes a novel, easy to implement methodological approach to refining study design that has the potential to improve reproducibility of animal-based research. [LA] Researchers will want to know that their animals are in good health. But how can you tell if your zebrafish are in pain or stressed out from a procedure? Recent studies say that such fish tend to be less active and linger near the bottoms of their tanks. Perhaps easy enough to gauge by eye if your numbers are small, but some facilities maintain hundreds of tanks at a time. To help make those assessments more feasible for large-scale operations, and to reduce the risk of human bias, researchers from Lynne Sneddon’s lab at the University of Liverpool developed the automated Fish Behaviour Index, or FBI. The FBI is based on swimming activity and swimming distance, recorded by cameras. The researchers developed the FBI with female AB strain zebrafish after different invasive procedures, such fin clipping and PIT tagging, and to evaluate the effects of different analgesics. Against human assessment, the FBI correlated well. You can read more and find links to the software in the journal Scientific Reports.

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The June episode of 3-Minute 3Rs from the North American 3Rs Collaborative (www.na3rsc.org), the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)Papers; 1. https://bit.ly/2UH4hZt2. https://go.nature.com/2W5CIea3. https://bit.ly/2Fi4td4[LA] On the road to the clinic, immunocompromised mice transplanted with patient-derived xenografts have been a frequent step for testing a drug’s efficacy and safety. However, there may soon be fisher options afoot that might help replace some of those mice. Writing in the June issue of the journal Cell, David Langenau and colleagues describe an adult zebrafish xenograft model, into which they successfully engrafted several different solid pediatric tumors. Larval versions had existed before, but an adult model offers a few notable advantages. Bigger fish can bear a larger tumor burden for a longer period of time than the small fry. Adults can be orally gavaged, meaning researchers can know exactly how much of a drug they are getting. And with acclimation, they can handle the heat – the Langenau lab kept them at 37 degrees Celsius, the same temperature a tumor will find in the human body. In the paper, the zebrafish model performed comparably to a murine one. As such, xenografted zebrafish might be valid alternatives to mice, at least at the early stages of drug testing. [NA3RsC] What if it were possible to screen for compounds with cardiotoxic side effects in less than a few hours? Dr. Farraj and colleagues from the National Health and Environmental Effects Research Laboratory explore the possibility of using zebrafish embryos to accurately predict cardiotoxicity quickly. In their publication in Scientific Reports, Farraj and colleagues describe a multi-well platform with the ability to automatically quantify the heart rate of multiple zebrafish embryos per imaging field using an algorithm they call FisHRateZ. To demonstrate the utility of this approach, embryos were exposed to compounds known to either increase or decrease the heart rate. FisHRateZ accurately detected expected changes in heart rate with high sensitivity. The whole process is also quite fast. The assessment of a full 96-well plate can be conducted in less than 50 minutes. One limitation, they note, is the inability of FisHRateZ to measure arrhythmias. Check out this paper to learn more about the platform and download the free video processing algorithms.[NC3Rs] Scientists often use indirect measures such as changes in behavior or physiology to assess whether the welfare of the animal has been compromised, but methods which have been applied to larger non-aquatic animals are often not appropriate for fish. Recently, a mathematical method has come to the rescue, allowing the amount a fish moves to be used as a marker for welfare. Dr Anthony Deakin and team at the University of Liverpool developed the method, which used cameras to track the movement of individually housed zebrafish before and after undergoing potentially painful procedures. The trajectories created by the movement of the fish were analysed using the fractal dimension technique, which is a method used to reduce the complexity of data and produce a simple scale on which to compare the adversity of different procedures. This revealed significant reductions in movement complexity in procedural groups in comparison to the control. In particular, fin-clipped and PIT-tagged groups showed reduced movement complexity, highlighting the likelihood that these routine procedures are indeed painful. The addition of lidocaine reduced the impact of the fin clipping, demonstrating that this analgesia is an advantageous refinement in fish. Follow the link in the description to find out more about this non-invasive tool to monitor zebrafish welfare.

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The May episode of 3-Minute 3Rs from the North American 3Rs Collaborative (www.na3rsc.org), the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)Papers:1. https://bit.ly/2vYuUPR2. https://go.nature.com/2w2tzre3. https://go.nature.com/2VEjEHz[NA3RC] Early mortality of pups in breeding colonies can reach upwards of 50% or higher depending upon the strain. While high pup mortality is often considered "normal", it instead may be indicative of an underestimated animal welfare issue that requires attention. Leidinger and colleagues investigate the influence of environmental enrichment as one way to mitigate early pup mortality rates. Enrichment, with nesting material and a shelter, protected against early mortality. Survival rates of pups born into enriched environments were more than double that of pups born into impoverished environments. The impoverished environment also had a negative impact on the developmental course of the remaining pups. Enrichment, among many potential benefits, may have reduced maternal stress and improved thermoregulation, which is particularly important for newborn pups. The findings suggest that early pup mortality is an animal welfare problem that can be improved through the characterization and selection of appropriate enrichment strategies. The refinement in breeding conditions can improve pup welfare and survival and lead to the need for smaller breeding colonies. [LA] Enrichment helps pups, check. But how can you tell how they’re doing in the first place? Invasive measures are challenging in such small animals, and pups lack features like fur and certain behaviors that can visually indicate health in adults. Dorothee Viemann and colleagues at Hannover Medical School recently proposed a new clinical health scoring system for neonatal mice to solve the pup assessment problem. They developed a score sheet based on observations of movement, nursing, and skin color and from quick examinations of capillary refill time, dehydration, reaction to tactile stimulus, and abdominal palpitation. They put their scoring to the test with healthy Black6 pup and two sepsis models known to cause mortality. The scoring correlated well with diseases, and was even more sensitive than blood-based biomarkers... [NC3Rs]…most creatures take great efforts to separate their ‘doings’ from anything else they do. Rodents especially will almost always have a dedicated latrine area away from their nest site. Laboratory mice, typically housed in small, open chambers are prevented from performing this instinctual behavior, and their nesting material is often found in close contact with their droppings. Joanna Makowska from the University of British Columbia and her colleagues have investigated how this housing arrangement affects the behavior of these animals. Groups of mice were kept in one of two housing systems. A standard single cage or a rather more luxurious series of three interconnected cages set up in a similar manner to rodent burrows in the wild. Mice in the triple cages were observed to segregate their nesting and latrine areas between different cages. Single cage mice attempted to separate these sites with their cage, urinating and defecating away from their nests. However the day to day movements of the animals invariably scattered the contents of the enclosure. Notably, mice housed in triple cages showed more affiliative behaviors, such as grooming or resting with one another than those in single enclosures. indicating a stronger social bond between the animals. They also were less disturbed by weekly cage cleaning, a typically stressful and highly disruptive event for lab mice. In the paper, published in Scientific Reports this April the authors conclude that mice find being housed in contact with their waste aversive, and that facilitating separation of nesting and latrine areas could be a simple, effective way of improving the welfare.

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This is the April episode of 3-Minute 3Rs, brought to you by the North American 3Rs Collaborative (www.na3rsc.org), the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)Source papers:1. Characterizing Sources of Variability in Zebrafish Embryo Screening Protocols. https://bit.ly/2VP8njP2. Effective chair training methods for neuroscience research involving rhesus macaques. https://bit.ly/2V7NOSv3. AutonoMouse. https://bit.ly/2VP8tbb. Video: https://www.youtube.com/watch?v=6d7ctvj953M[NA3RsC] The zebrafish embryo, considered to experience little or no pain or distress, has been investigated as a humane replacement to the adult fish and adopted for chemical characterization and hazard assessments. The Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative was developed by the NTP NICEATM to address inconsistent test guidelines and identify sources of variability for zebrafish embryo-based assays. The goals included gathering input from zebrafish investigators on sources of outcome variability and facilitating a multi-laboratory evaluation of protocol variables. The SEAZIT team identified key factors producing variability which are highlighted in a report in a recent publication in ALTEX. While the current effort focused on defining a set of parameters that all researchers should consider, further advancements would improve the ease with which zebrafish embryo screening data could be used to inform a broad range of health-related research areas, including chemical hazard assessments. As the SEAZIT effort progresses, connections continue to be made with zebrafish researchers to collect a broad range of opinions and foster greater consistency in experimental approaches. [LA]... To move an animal as large as a macaque, many primate labs use special chair-like devices in which the animals are restrained. It can take considerable time to gets them trained and comfortable in the chairs, and the process can be stressful to all the primates involved. A team at Oxford, which uses macaques for neuroscience research, set out to refine their transportation training procedures. Details about their approach were published in the Journal of Neuroscience Methods at the beginning of the month. In short summary, they found that short but frequent training sessions started soon after animals arrived at the facility and using mostly positive re-enforcement in the form of tasty treats was most effective. They recommend that animals be trained in pairs or small groups rather than alone, and that negative reinforcement be limited as much as possible. They found that they could get their macaques into their transport devices in as few as 10 training sessions. [NC3Rs] Behavioral tasks in rodents have been an important tool in neuroscience research, but they are not without limitations. Traditional approaches are labor-intensive and require handling of the animals to move them to the testing arena, which can cause stress and affect the experimental outcomes. Erskine and colleagues...developed a fully automated system that addresses these issues. AutonoMouse... houses cohorts of up to 25 mice and allows continuous training and testing in enriched home cage environment, over a period of several months. Controlled by an ID microchip in each mouse, the cage is designed so that the animals can enter a behavioral test area individually, access water and engage in the tasks whenever they chose. The automated weighing module can enable monitoring to make sure the mice receive the amount of fluid they require. The system is also designed for minimal disturbance during cleaning, including bedding exchange without direct human contact. Detailed instructions and software are included in the paper to facilitate setup in other labs. The technology is less time-consuming and offers benefits to animal welfare, as the animals are kept in their home cage and don’t have to be picked up as often.

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This is the March episode of 3-Minute 3Rs, brought to you by the North American 3Rs Collaborative (www.na3rsc.org, the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)The papers behind the pod:1. Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS. https://bit.ly/2uiF64X2. A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery. https://go.nature.com/2Cu7u8C3. No-touch measurements of vital signs in small conscious animals. https://bit.ly/2WdN5Mw[NC3Rs] A team led by Dr Rickie Patani has developed a human-derived model of ALS that could bring us a step closer to treating the disease effectively while avoiding the use of animals altogether.Animal models are widely used to study ALS, also known as motor neurone disease, but current therapies can only slow its progression – and even then, the effect is modest. Instead, Dr Patani's team, based at UCL and the Francis Crick Institute, used human induced pluripotent stem cells to study how ALS causes motor neurones to degenerate. They investigated the molecular processes that lead to the death of motor neurones, which are kickstarted by the loss of a protein called TDP-43 from the cell nucleus. They also discovered that ALS makes astrocytes degenerate too, so they can’t play their usual role in helping motor neurones survive, compounding the effects of the disease. For this work, Dr Patani was awarded the NC3Rs’ International 3Rs Prize earlier this month. The prize is sponsored by GSK and celebrates outstanding 3Rs science every year. Read the paper in Cell Reports or visit the NC3Rs website to learn more about the 3Rs Prize. [LA] Rodents remain popular for in vivo validation of novel drugs. But screening candidates is costly to do in rodents, which has researchers looking for alternatives to evaluate compounds in a higher throughput manner. Zebrafish are increasingly used for such screening purposes. A new paper from Richard van Rijn’s lab at Purdue published in the journal Scientific Reports evaluates a zebrafish screen for drugs that Transient Receptor Potential A1, or TRPA1. TRPA1 encodes a calcium ion channel and has been shown to be involved in pain perception in rodent models. In zebrafish, activating TRPA1 causes hyperlocomotion, which the researchers hypothesized could be a useful phenotypic readout of drug efficacy. They tested compounds known to activate and inactivate TRPA1 in human cells, mice, and zebrafish larvae and found that the compounds affect all three models in a dose-dependent manner. Evaluation can be somewhat tricky in the zebrafish because they have a second copy of TRPA1 to contend with, but the fish could still help screen initial compounds before researchers take them onward. [LA] What if you could measure the vital signs of your animals without having to prep or handle them? Engineers at Cornell recently described the use of radio frequency near-field coherent sensing to do just that in a paper published in Science Advances. They developed the technology first for humans, but have now shown its potential for use with small animals in real-time. The technology uses radio waves. These penetrate the body and can be used to detect the motion of internal organs. When the signal is processed, parameters like heart rate and respiration rate can be captured. The system can be wired or wireless, and was shown to work with an anesthetized rat and freely moving hamster, Russian tortoise, and betta fish. There was some variability and further comparisons with existing methods would help clarify the accuracy and robustness of the new system, but near-field coherent sensing could be a promising new way to keep an eye on animal vital signs.

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This is the February episode of 3-Minute 3Rs, brought to you by the North American 3Rs Collaborative (www.na3rsc.org, the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)The papers behind the pod:1. 3D-printed Wash Station with Integrated Anesthesia Delivery Manifold for High-throughput Depilation of Laboratory Mice. https://bit.ly/2IpGrAP2. DeepSqueak: a deep learning-based system for detection and analysis of ultrasonic vocalizations .https://go.nature.com/2tupSJK3. Super-Mendelian inheritance mediated by CRISPR–Cas9 in the female mouse germline. https://go.nature.com/2RgnoYw[NA3RsC] Depilation (hair removal) is needed for many procedures done with laboratory mice, including surgery and imaging. Commercial depilatory creams are a safe, efficacious, and often-used method to achieve this hair removal. However, the use of these creams tends to be messy and time consuming, and wide procedural variation exists between laboratories. Furthermore, mice are generally under anesthesia during depilation, so increasing efficiency of the process is a valuable refinement to minimize anesthesia-associated adverse effects. Researchers from the University of Notre Dame have designed a 3D-printed device to simplify, standardize, and streamline the hair removal process. The Mouse Depilation Station (MDS) consists of an elevated stage with flow-through slats and is designed for integration with a self-scavenging anesthesia manifold and waste collection tray. It allows three mice to be depilated in parallel, thus improving efficiency compared to processing mice in sequence. The design files for the MDS stage are available online for free download – read the full article in JAALAS for the link.[NC3Rs] Imagine if laboratory animals could tell us how they were feeling. Scientists at the University of Washington in Seattle have moved this a step closer by developing Deepsqueak, a system to decipher rodent ultrasonic vocalisations. Usually outside the human range of hearing, rats and mice use a characteristic set of squeaks to communicate with one another. Writing in the journal Neuropsychopharmacology Coffey et al have used deep learning algorithms similar to those which power Alexa’s speech recognition abilities to identify and categorise rodent ‘syllables’. The rodent chatter can be linked to behaviours and physiological parameters, allowing an electronic dictionary of rodent calls to be built. One particular area of interest is in neuroscience research, where rodent behavioural tests are routinely applied to study a diverse collection of outcomes and diseases.By using fully automated monitoring of ultrasonic vocalisations during experiments an additional level of behavioural information can be collected, providing a non-invasive method for monitoring rodent welfare. [LA] All animals inherit their genes from their parents, but there’s a 50/50 chance whether a particular copy of a gene will come from the mother or from the father. When you want to make a mouse model of a particular genetic mutation, that often means a lot of animals are bred that don’t have the desired change. But what if you could weigh that probability? It’s been demonstrated already in insects, but Kimberly Cooper from the University of California San Diego and her colleagues bring active genetics to mammals for the first time in a new publication in Nature. They developed a CRISPR/Cas9 construct that could produce of litter of mice in which over 70% of the pups had the desired mutation. It only worked in females so far, but the approach proves that it is feasible to weight the genetic coin in mice.

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This is the January episode of 3-Minute 3Rs, brought to you by the North American 3Rs Collaborative (www.na3rsc.org, the NC3Rs (www.nc3rs.org.uk), and Lab Animal (www.nature.com/laban)The papers behind the pod:1. An Atypical Parvovirus Drives Chronic Tubulointerstitial Nephropathy and Kidney Fibrosis https://bit.ly/2FA6dPA2. Remote Welfare Monitoring of Rodents Using Thermal Imaging https://bit.ly/2suGlgm3. No experimental evidence of stress-induced hyperthermia in zebrafish (Danio rerio)https://bit.ly/2Db8m2P[NA3RsC] Chronic kidney disease results from fibrosis of the kidney parenchyma due to a variety of inciting factors. Existing models of tubulointerstitial nephropathy and fibrosis involves administration of nephrotoxic compounds or surgical interventions, but these models often fail to recapitulate the chronic, insidious nature of fibrotic disease. In a recent publication from the October 4, 2018 issue of Cell, Ben Roediger and his colleagues at the University of Sidney Australia observed a high incidence of kidney disease in 3 separate colonies of immunodeficient mice; disease was characterized by enlarged nuclei in tubular epithelial cells in addition to intranuclear inclusions and marginated chromatin. RNA sequencing identified a complete novel viral genome that was given the name ‘mouse kidney parvovirus.’ Resulting pathology included extensive fibrotic changes to the kidneys with significantly reduced renal mass. Virus was absent in mouse colonies free of kidney disease, transmissible following co-housing and infected mice exhibited similar transcriptional changes to humans with fibrotic disease. Ultimately, mouse kidney parvovirus may represent a new tool for understanding viral nephropathy as well as the fibrotic processes and pathogenesis of chronic kidney disease.[NC3RS] To assess the well-being of an animal, it is useful to be able to continuously monitor their vital signs, such as heart rate and breathing rate, and the amount they move. Vital signs can be assessed by implanting a telemetry sensor or using electrocardiography. However, these methods can lead to unnecessary discomfort and stress for the animal and can compromise their welfare. A recent study by Pereira et al. addresses these issues by using thermal imaging to monitor animal welfare. Thermal imaging is a remote and passive monitoring technique that can continuously record the heat emitted from the animal’s body. Using this technology, the researchers were able to remotely monitor breathing rate of anaesthetised rats and mice, and the position and velocity of active rodents during open field tests. This promising technique has the potential to refine current monitoring practice and improve animal welfare. Follow the link in the description to find out more.[LA] When endothermic animals, like ourselves or our laboratory mammals, get stressed, we often get hot. Literally. But not all animals control their internal body temperature. A 2015 paper published in the Proceedings of the Royal Society B indicated that zebrafish stressed out from temporary confinement will deliberately move to a warmer area of their tank in order to raise their body temperature. This suggests that they too exhibit stress-induced hyperthermia. But questions have lingered about whether the fish were actively seeking heat or rather, just moving away from the chamber where they had been confined. Writing in the Journal of Experimental Biology, researchers from the University of St. Andrews repeated that experiment, but included control tanks without a temperature gradient. They saw no discernible effect. They also developed a second tank set up - in that case, stressed fish actually spent a little less time in warmer water. Don’t neglect zebrafish stress, the authors stress, but their results suggest that the fish don’t experience stress-induced hyperthermia in the same as other animals.

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