3 minute 3Rs, brought to you by Lab Animal at Nature Research and the NC3Rs in London, is a quick monthly podcast summarizing the latest research and news in 3Rs Science and Technology. This month, we look at rodent big brother for 24/7 remote monitoring of rodent behavior, the alliance of genome resources, and the experimental design assistant for improving the rigor and reproducibility of animal research.

3 Minute 3Rs November

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/2FA6dPA
2. Remote Welfare Monitoring of Rodents Using Thermal Imaging https://bit.ly/2suGlgm
3. 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.

3 Minute 3Rs January 2019

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/2UH4hZt
2. https://go.nature.com/2W5CIea
3. 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.

NovaLibrary

3 Minute 3Rs June 2019

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/eaav6528
2. https://www.nature.com/articles/s41598-019-44705-2
3. 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.

3 Minute 3Rs July 2019

The August episode of 3-Minute 3Rs.

Papers:
1. https://advances.sciencemag.org/content/5/7/eaaw4099
2. https://www.ncbi.nlm.nih.gov/pubmed/31026040
3. 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.

3 Minute 3Rs August 2019

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.long
2. https://www.sciencedirect.com/science/article/pii/S001216061830006X
3. 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.

3 Minute 3Rs September 2019

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).

Papers
1. Modelling human hepato-biliary-pancreatic organogenesis from the foregut–midgut boundary
https://www.nature.com/articles/s41586-019-1598-0
2. Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms https://www.nature.com/articles/s41598-019-50920-8
3. Analysis of vertebrate vision in a 384-well imaging system. Thorn RJ. Sci Rep. https://www.ncbi.nlm.nih.gov/pubmed/31562366

Transcript
[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.

3 Minute 3Rs October 2019

The November 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:
1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662399/
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371849/
3. https://pubs.rsc.org/en/content/articlelanding/2019/LC/C9LC00160C 

[NA3RsC] Evaluation of developmental and reproductive toxicity is a key aspect of drug development. Each year, 5-7% of newborns worldwide are born with serious birth defects, a number correlated to increased use of pharmaceutical drugs for managing health conditions. Reproductive toxicology studies account for roughly 90% of all animal use in toxicological safety assessment. The FDA requires that these animal studies be performed in two species, often rodents and rabbits. The embryonic stem cell test (EST) is an alternative in vitro assay that does not require the use of pregnant animals but is not yet powerful enough to provide a complete alternative, as it only considers the direct toxicity of compounds. Consequently, in vivo animal models are still required to evaluate effects on drug metabolism.
This research established an in vitro platform (metaEST) which uses 3D embryoid bodies and 3D primary human liver microtissues on a chip to evaluate alterations in metabolism. Not only does this significantly reduce the number of animals required, these results demonstrate the platform’s ability to evaluate metabolic effects that are missed using the standard EST assay and mimic physiologically relevant conditions in vitro.

[NC3Rs] Botulinum neurotoxin can cause fatal paralysis following ingestion or infection of a wound and is a potential bioterrorism agent. Therefore, the ability to quickly and accurately detect the toxin in a sample is crucial. The current method used to detect the toxin is the mouse lethality bioassay, where mice are injected with neurotoxin to determine the LD50 value, or the dose that kills half the animals. This uses approximately 70,000 mice per year in the UK and more than 600,000 mice annually worldwide and causes severe suffering prior to death. Researchers at the Centers for Disease Control and Prevention in the US have developed a mass spectrometry-based method that can successfully identify botulinum neurotoxin activity for the four human-toxic serotypes. It is faster - the test can be done in just 4- 6 hours on the spectrometer rather than the 1- 4 days typically required by the mouse bioassay. The test also requires samples with volumes as low as 100 µL per toxin type, instead of the 1 mL required for the mouse bioassay, avoiding the problem of insufficient sample volumes. This mass spectrometry- based assay is a fast and accurate alternative to the mouse bioassay and further implementation of the technique has the potential to reduce the number of animals used for this purpose. 


[LA] Tissues do not exist in isolation from one another. As increasingly sophisticated organ-on-chip technologies continue to emerge, many researchers want to link those chips together to mimic multi-organ systems. That’s been easier said than done. Yi-Chin however has been working on making better connections. The new microfluidic platform she helped developed consists of modular tissue and fluid control units, referred to as TILE modules. With the help of magnetic connectors, these modules can be arranged and re-arranged into different perfusion circuits as needed. It’s backwards compatible too, meaning existing microfluidic devices can be used. The team involved demonstrated the platform in action with 2 and 3 tissue modules that mimic liver-mediated bio activation in response to a nutraceutical and a cancer prodrug. All using human cells, rather than animal models.

3 Minute 3Rs November 2019

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).

Papers: 
1. Cage aggression in group-housed laboratory male mice: an international data crowdsourcing project. https://www.nature.com/articles/s41598-019-51674-z
2. Towards animal-free neurotoxicity screening: Applicability of hiPSC-derived neuronal models for in vitro seizure liability assessmenthttps://www.altex.org/index.php/altex/article/view/1321
3. Optimal solid state neurons https://www.nature.com/articles/s41467-019-13177-3


[NC3Rs] Mice are social creatures. It's why we house them in groups in the lab to improve their welfare and allow the animals to make social interactions that simply aren't possible if they're individually housed. However, the social groupings mice make in the wild are complex, with hierarchies of social dominance. In the lab group has mice are kept in cages, which means dominant and subordinate males must stay in close quarters, which can lead to aggression related stress and injuries. To better understand this welfare concern, the NC3Rs have used a crowdsourcing approach, engaging with lab technicians worldwide to estimate the prevalence of male mouse aggression. Data from over 100,000 mice in over 45,000 cages was submitted for analysis, which has been published last month in Scientific Reports.The publication by Lidster et al. recommends small practical husbandry changes such as spot cleaning cages or transferring nesting material to minimize male mouse aggression. You can find out more in the link in the description.

 [NA3RsC] While drug approval rates are on the rise, only about 10% of drugs in phase 1 development will gain full approval. One of the causes of drug attrition is drug-induced seizures; There is a need for more translational in vitro alternatives to predict seizure liability. Here, the authors evaluated three human induced pluripotent stem cell (hiPSC)-derived neuronal models. All models demonstrated development of functional neuronal networks that exhibited parameters linked to seizure-like events in vivo. These parameters were then modulated with three compounds known to induce seizures. Differences in sensitivity were found between the models. Yet, compared to rat primary cortical neurons, the in vitro models were equally, or better able to detect seizureogenicity. While the authors recommend more compounds be tested and the available models further characterized before serving as full replacement, this data indicates the potential of these in vitro models as initial screening tools for seizure liability assessment which could lead to reduced animal use requirements. 

[LA] Neurons transmit electrical signals – so too do silicon chips. Moving that in vivo function in silico has long been a goal of many neuroscientists, but biological neurons are complex, and their electrical properties and nuances have been difficult to predict and mimic artificially. That hasn’t stopped researchers from trying. A team led by Alain Nogaret at the University of Bath recently made progress, presenting artificial neurons on a silicon chip that matched the patterns of rat neurons with over 90% accuracy. It took lots of mathematical modeling, derived equations, and tweaking, but the team was able to optimize the chips so that they relayed electrical signals in the non-linear fashion that’s characteristic of biological neurons, all with considerably less power required than prior attempts. The design details and rat neuron comparisons can be found in the journal Nature Communications.

3 Minute 3Rs December 2019

The January 2020 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:
1.  https://doi.org/10.15252/embr.201949709
2. https://www.nature.com/articles/s41598-019-56860-7
3. https://doi.org/10.1177/0023677219879455

[NC3Rs] Academic scientists produce many of the clever ideas which end up as medicines. However, the results in the papers reporting the work are almost always positive, and null or negative results rarely get a look in – a phenomenon known as publication bias. This is problematic as the negative or null data could be useful to other researchers. It can also lead to a culture of striving for positive results, potentially increasing poor experimental design or the inappropriate use of statistics. Overall this contributes to the so-called ‘reproducibility crisis’ and means that animals are used in studies that are not correctly designed to demonstrate an effect. In response to this, scientists at the German Centre for the Protection of Laboratory Animals have set up a global ‘Animal Studies Registry’ where a researcher can preregister their study and following completion the Registry is updated with the results. This publicly accessible database allows scrutiny of the planning and execution of animal studies. The Registry could become an important addition to the preregistration landscape - time will demonstrate the impact this can have on reproducibility and the use of animals in research. You can read more about the registry in this month’s EMBO Reports via the link in the description. 

[LA] Next, how do you handle your mice? There’s growing evidence that picking up the animals with cupped hands or a tunnel is less aversive than picking them up by the tail, but mice still need to be familiarized with the process. Across an entire animal facility, that time can add up. What’s practical? Kelly Gouveia and Jane Hurst from the University of Liverpool have some suggestions.  They recently evaluated BALBc mice handled by the tail, cupped hands, or a tunnel in three experiments: the first looked at the impact of holding duration; the second, the frequency of handling; and the third, responses to repeated immobilization for necessities such as subcutaneous injections. Tunnels rose to the top. Mice became familiar with tunnel handling from use during routine animal husbandry – a couple seconds each cage change was sufficient, and tunnel-acclimated animals remained less stressed than their tail-handled peers, even after the repeated immobilizations,.  For all the details, see the publication in the Journal Scientific Reports. 
And finally, let’s hit the wheel running with another refinement. 
[NA3RsC] Objectively measuring well-being in laboratory mice can be challenging. In a study published in the journal Laboratory Animals, voluntary wheel running behavior was evaluated as a measure of discomfort and disease severity in a mouse model of human Inflammatory Bowel Disease. While voluntary wheel running has been successfully used to assess severity levels in singly housed mice, mice are social animals. Here, voluntary wheel running was assessed in group housed mice undergoing DSS-induced colitis and in group housed mice that underwent a blood collection procedure at 3 different time points throughout the study. While a there was a significant reduction in wheel running and body weight, and a significant elevation in histological scores as disease severity increased, there were few signs of disturbed well-being based on clinical scores alone. Wheel running was also decreased in mice that underwent blood collection while other measures remained unaffected. This study suggests that voluntary wheel running may be a sensitive and suitable tool for individual severity assessment in group housed mice and offers a refinement to welfare by enabling social housing

3 Minute 3rs January 2020

The February 2020 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).

1. https://www.sciencedirect.com/science/article/pii/S0092867419313236
2. https://www.sciencedirect.com/science/article/pii/S0165027019303097
3. https://www.ncbi.nlm.nih.gov/pubmed/31852563

[NC3Rs] If you are bitten by a snake today, your anti venom treatment would be largely similar to if you were bitten 100 years ago.
Anti venom is created by isolating antibodies from a large animal, typically a horse, after the animal has been given repeated injections of small amounts of venom. But due to issues with anti venom production, efficacy and safety standards, over 100,000 people worldwide will die from snake bites each year. Venom is a complex mix of molecules, which vary between different snake species and can only be obtained by milking snakes. This has been an obstacle in anti venom research. But now scientists from the Netherlands have generated an in vitro method for producing venom from snake venom gland cells cultured in 3d structures. In a publication Cell, Post et al. successfully cultured snake venom gland organoids from nine different snake species including the Cape Coral Cobra.
The organoids growing definitely and produce functioning toxins, which could be used to further anti venom research. The organoids could also have further reaching consequences by enabling the development of new treatment options that might avoid the need for animals altogether. You can read more about snake venom gland organoids by following the link in the description.

[LA] Neurons are often studied in cell cultures, but those cultures have to come from an animal originally. Nor do they last all that long – primary neuron cultures generally need to be used in a few weeks. That can put a rush on reproducing experiments, and often means extra animals need to be maintained for future use.  
To make cultures from a given cohort of mice last, researchers from University College London present a protocol to cryopreserve brain tissue and prepare neuronal cultures from it.  Neurons retrieved from cryopreserved mouse hippocampi perform on par with freshly dissected cultures, with most physiological properties appearing unaffected by the process. Properly stored in liquid nitrogen, tissues can be frozen for at least two years. 
The results and step-by-step details can be found in the Journal of Neuroscience Methods.  


[NA3RsC] In a manuscript recently published in JAALAS, Dr. Brianna Gaskill and Dr. Joe Garner address considerations for publishing negative results and make practical recommendations for conducting sufficiently powered research. This includes challenging the traditional use of a priori power analyses and pilot studies for justifications is animal numbers and instead using more sophisticated experimental designs and calculating the least significant number of experimental units necessary to produce sufficient power. The authors suggest that researchers collaborate with experts in biostatistics, comparative medicine and other disciplines early in the research planning process in order to consider improvements in experimental design and select of appropriate statistical tests. Such collaboration will ensure the creation of high-quality experiments, helping researchers to address the 3Rs while improving reproducibility and translational value of animal studies. Finally, the authors offer recommendations for reporting to improve the interpretation of results.

3 Minute 3Rs February 2020

The March 2020 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:
1. https://pubmed.ncbi.nlm.nih.gov/32076633
2. https://www.sciencedirect.com/science/article/pii/S0273230020300507
3. https://www.nature.com/articles/s41598-020-59012-4

Transcript
[NC3Rs] Nanomedicines have many potential advantages, including their ability to home to specific diseased tissues and exert a localised effect. Due to their relatively large size, the biodistribution of nanomedicines does not follow the same rules as small molecules and is  largely dictated by the diameter of the blood vessels they pass through en route to their target tissue. This means that although current methods to assess biodistribution rely heavily on the use of animals including rodents and non-human primates, the results can vary greatly between preclinical species and man. Using an in vitro cell based model of nanoparticle uptake to build a computer simulation, Edward Price and colleagues at the University of Central Florida have developed a predictive system to replace animal use for determining nanomedicine PKPD. The model takes account of blood vessel size, tissue-specific porosity and the kinetics of distribution to cellular and paracellular compartments. By being able to better predict where nanomedicines will end up in the body, scientists can design safer and more effective drugs based on nanomaterials without using animals to assess biodistribution. For more information on their study, see the paper linked in the podcast description.

[NA3RsC] Human safety is a key priority when developing new drugs. Toxicity testing often requires drugs to be used in animal models before human clinical trials. Historically, toxicology testing has involved two species: one rodent and one non-rodent. However, in some cases, only 1 animal species is used successfully. This month, an international working group of 37 diverse organizations led by Dr. Helen Prior published a paper on this topic in Regulatory Toxicology and Pharmacology. They reviewed data from 172 potential new drugs, across a wide range of small molecules and vaccines. These potential new drugs were tested in either 1 or 2 species. The working group found that often when 2 species were used they showed similar toxicity profiles in both. Therefore, it may be possible to reduce the number of species new drugs are tested in while maintaining human safety. 

[LA]. Social housing is generally recommended for social species, such as the mouse, whenever possible. But housing animals together can lead to incidences of aggression – an unwanted outcome for the welfare of the animals involved. Female mice tend to get along just fine, but males can sometimes be another story.  In a new 14 week study measuring aggression and physical signs of well-being in male Cd-1 mice, a stock with an aggressive reputation,  Paulin Jirkof and colleagues consider three variables that might influence how well those animals get along: group size, the age at which males are allocated to their groups, and handling frequency.  The full results can be found in the journal Scientific Reports but overall levels of aggression were actually low. And for these mice, three was not a crowd.  Trios got on better than pairs, with solo males scoring the lowest on measures of well-being.

3 Minute 3Rs March 2020

Behind the scenes with the authors and experts shaping in vivo science, technology and policy

LabAnimal

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