Norecopa: A global knowledge base of resources for improving animal research and testing.

There are good ethical, legal and scientific reasons for ensuring that our use of animals in research and testing is limited to the lowest number of animals, and that those which are used are treated as humanely as possible, while at the same time providing reliable, reproducible and translatable data which is adequately reported. Unfortunately, there is widespread evidence that there is room for improvement in all these areas. This paper describes the Norecopa website, which offers links to global resources which can be used to resolve these issues. Much of the website content is linked to the PREPARE guidelines for planning any research or testing which appears to need animals. Attention to detail on all steps of the pathway from early planning to manuscript submission should lead to better science, improved animal welfare, and fewer health and safety accidents. This will also minimize the chances of manuscript rejection due to inadequate planning, avoiding a waste of human resources and animal lives.

Guidelines for planning and conducting high-quality research and testing on animals.

There are important scientific, legal and ethical reasons for optimising the quality of animal research and testing. Concerns about the reproducibility and translatability of animal studies are now being voiced not only by those opposed to animal use, but also by scientists themselves. Many of the attempts to improve reproducibility have, until recently, focused on ways in which the reporting of animal studies can be improved. Many reporting guidelines have been written. Better reporting cannot, however, improve the quality of work that has already been carried out - for this purpose better planning is required. Planning animal studies should involve close collaboration with the animal facility where the work is to be performed, from as early a stage as possible. In this way, weaknesses in the protocol will be detected and changes can be made before it is too late. Improved planning must focus on more than the "mathematical" elements of experimental design such as randomisation, blinding and statistical methods. This should include focus on practical details such as the standard of the facility, any need for education and training, and all the factors which can improve animal welfare. The PREPARE (Planning Research and Experimental Procedures on Animals: Recommendations for Excellence) checklist was developed to help scientists be more aware of all the issues which may affect their experiments. The checklist is supported by comprehensive webpages containing more information, with links to the latest resources that have been developed for each topic on the list.

The Role of the Three Rs in Improving the Planning and Reproducibility of Animal Experiments.

Training in the design of animal experiments focuses all too often on those aspects which can be approached mathematically, such as the number of animals needed to deliver a robust result, allocation of group size, and techniques such as randomization, blocking and statistical analysis. Important as they are, these are only a small part of the process of planning animal experiments. Additional key elements include refinements of housing, husbandry and procedures, health and safety, and attention at all stages to animal welfare. Advances in technology and laboratory animal science have led to improvements in care and husbandry, better provision of anesthetics and analgesics, refined methods of drug administration, greater competence in welfare assessment and application of humane endpoints. These improvements require continual dialogue between scientists, facility managers and technical staff, a practice that is a key feature of what has become known as the culture of care. This embodies a commitment to improving animal welfare, scientific quality, staff care and transparency for all stakeholders. Attention to both the physical and mental health of all those directly or indirectly involved in animal research is now an important part of the process of planning and conducting animal experiments. Efforts during the last 30 years to increase the internal and external validity of animal experiments have tended to concentrate on the production of guidelines to improve the quality of reporting animal experiments, rather than for planning them. Recently, comprehensive guidelines for planning animal studies have been published, to redress this imbalance. These will be described in this paper. Endorsement of this overarching influence of the Three R concept, by all the stakeholders, will not only reduce animal numbers and improve animal welfare, but also lead to more reliable and reproducible research which should improve translation of pre-clinical studies into tangible clinical benefit.

PREPARE: guidelines for planning animal research and testing.

There is widespread concern about the quality, reproducibility and translatability of studies involving research animals. Although there are a number of reporting guidelines available, there is very little overarching guidance on how to plan animal experiments, despite the fact that this is the logical place to start ensuring quality. In this paper we present the PREPARE guidelines: Planning Research and Experimental Procedures on Animals: Recommendations for Excellence. PREPARE covers the three broad areas which determine the quality of the preparation for animal studies: formulation, dialogue between scientists and the animal facility, and quality control of the various components in the study. Some topics overlap and the PREPARE checklist should be adapted to suit specific needs, for example in field research. Advice on use of the checklist is available on the Norecopa website, with links to guidelines for animal research and testing, at https://norecopa.no/PREPARE .

Zika virus tropism and interactions in myelinating neural cell cultures: CNS cells and myelin are preferentially affected.

The recent global outbreak of Zika virus (ZIKV) infection has been linked to severe neurological disorders affecting the peripheral and central nervous systems (PNS and CNS, respectively). The pathobiology underlying these diverse clinical phenotypes are the subject of intense research; however, even the principal neural cell types vulnerable to productive Zika infection remain poorly characterised. Here we used CNS and PNS myelinating cultures from wild type and Ifnar1 knockout mice to examine neuronal and glial tropism and short-term consequences of direct infection with a Brazilian variant of ZIKV. Cell cultures were infected pre- or post-myelination for various intervals, then stained with cell-type and ZIKV-specific antibodies. In bypassing systemic immunity using ex vivo culture, and the type I interferon response in Ifnar1 deficient cells, we were able to evaluate the intrinsic infectivity of neural cells. Through systematic quantification of ZIKV infected cells in myelinating cultures, we found that ZIKV infection is enhanced in the absence of the type I interferon responses and that CNS cells are considerably more susceptible to infection than PNS cells. In particular, we demonstrate that CNS axons and myelinating oligodendrocytes are especially vulnerable to injury. These results have implications for understanding the pathobiology of neurological symptoms associated with ZIKV infection. Furthermore, we provide a quantifiable ex vivo infection model that can be used for fundamental and therapeutic studies on viral neuroinvasion and its consequences.

Good Science, Good Sense and Good Sensibilities: The Three Ss of Carol Newton.

The Three Rs principle of Replacement, Reduction and Refinement developed by William M. S. Russell and Rex L. Burch in the 1950s has achieved worldwide recognition as a means of reducing the impact of science on animals and improving their welfare. However, application of the Three Rs is still far from universal, and evidence-based methods to implement the Three Rs are still lacking in many areas of laboratory animal science. The purpose of this paper is to create interest in a less well-known but equally useful principle that complements the Three Rs, which was proposed by the American biomathematician Carol M. Newton in the 1970s: the Three Ss-Good Science, Good Sense and Good Sensibilities.

The development of response surface pathway design to reduce animal numbers in toxicity studies.

BACKGROUND: This study describes the development of Response Surface Pathway (RSP) design, assesses its performance and effectiveness in estimating LD50, and compares RSP with Up and Down Procedures (UDPs) and Random Walk (RW) design. METHODS: A basic 4-level RSP design was used on 36 male ICR mice given intraperitoneal doses of Yessotoxin. Simulations were performed to optimise the design. A k-adjustment factor was introduced to ensure coverage of the dose window and calculate the dose steps. Instead of using equal numbers of mice on all levels, the number of mice was increased at each design level. Additionally, the binomial outcome variable was changed to multinomial. The performance of the RSP designs and a comparison of UDPs and RW were assessed by simulations. The optimised 4-level RSP design was used on 24 female NMRI mice given Azaspiracid-1 intraperitoneally. RESULTS: The in vivo experiment with basic 4-level RSP design estimated the LD50 of Yessotoxin to be 463 µg/kgBW (95% CI: 383-535). By inclusion of the k-adjustment factor with equal or increasing numbers of mice on increasing dose levels, the estimate changed to 481 µg/kgBW (95% CI: 362-566) and 447 µg/kgBW (95% CI: 378-504 µg/kgBW), respectively. The optimised 4-level RSP estimated the LD50 to be 473 µg/kgBW (95% CI: 442-517). A similar increase in power was demonstrated using the optimised RSP design on real Azaspiracid-1 data. The simulations showed that the inclusion of the k-adjustment factor, reduction in sample size by increasing the number of mice on higher design levels and incorporation of a multinomial outcome gave estimates of the LD50 that were as good as those with the basic RSP design. Furthermore, optimised RSP design performed on just three levels reduced the number of animals from 36 to 15 without loss of information, when compared with the 4-level designs. Simulated comparison of the RSP design with UDPs and RW design demonstrated the superiority of RSP. CONCLUSION: Optimised RSP design reduces the number of animals needed. The design converges rapidly on the area of interest and is at least as efficient as both the UDPs and RW design.

Reduced LPA expression after peroxisome proliferator-activated receptor alpha (PPARalpha) activation in LPA-YAC transgenic mice.

BACKGROUND:: Apolipoprotein(a) (apo(a)), which is part of the atherogenic lipoprotein Lp(a), shares structural homology with plasminogen (plg). Genes coding for plasminogen (PLG) and apo(a) (LPA) are linked and situated 40kb apart in the telomeric region of the long arm of chromosome 6. LPA is naturally expressed only in primates and hedgehogs. Thus, access to knowledge regarding the mechanism by which LPA expression is regulated is limited due to shortage of appropriate animal models. However, mice transgenic for the human LPA gene have been produced. Lp(a) levels in man are genetically determined and not altered significantly by dietary changes. In contrast, mice transgenic for LPA-yeast artificial chromosome (LPA-YAC) have markedly reduced apo(a) levels after maintenance on a high-fat diet. LPA-YAC carries the 40kb LPA-PLG intergenic region, which includes a putative binding site for peroxisome proliferator-activated receptor alpha (PPARalpha). Therefore, we examined if fibrates, which exert their effect via PPARalpha, could alter LPA expression in transgenic mice. METHODS:: Two LPA transgenic mouse lines with or without the LPA-PLG intergenic region we fed either PPARalpha agonist fenofibrate (FF) or 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY 14643) containing diets for 3 weeks. For the study of serum apo(a) levels, blood were sampled prior the experiment and when the animals were sacrificed. For the study of gene expression pattern pieces of livers were collected and submerged in RNAlater buffer and stored at -70 degrees C until analysis by quantitative PCR. RESULTS AND CONCLUSIONS:: The results showed that fibrates reduce LPA expression in LPA-YAC transgenic mice, but have no impact on hepatic apo(a) mRNA or serum apo(a) protein levels in LPA-cDNA transgenic mice, which lack the LPA-PLG intergenic region. This suggests that the effect of fibrates on LPA expression is mediated upstream of the LPA gene. However, on the basis of current data it is not possible to conclude that PPARalpha is the primary factor that represses LPA expression in LPA-YAC transgenic mice. Negative correlation between FXR and apo(a) mRNA levels, in addition to putative FXR DNA binding sequence in LPA-PLG intergenic region, suggest that it is equally likely that reduced expression of LPA could be a secondary consequence of PPARalpha activation on other genes, such as FXR.

Effects of PCB99 and PCB153 exposure on spermatogenesis in young adult C57BL6 mice.

This study examined the effects of acute exposure to PCB99 (2,2',4,4',5-pentachlorobiphenyl), and PCB153 (2,2',4,4'5,5'-hexachlorobiphenyl), on spermatogenesis in 8-week-old C57BL6 mice. The mice were randomly allocated to PCB99 and PCB153 and a single dose of respectively 10 and 100 mg/kg was given by oral gavage. During the 6-week experiment, six mice per treatment group were sacrificed weekly, body weights were recorded and samples with respect to the male reproductive system were collected until further analysis. None of the treatments, showed changes in body weight or reproductive endpoints. Flow cytometric analysis revealed spermatogenesis to be unaffected. However, PCB99 and PCB153 showed a significant increase in Leydig cell apoptosis. The results from the present study indicate that the male reproductive system is relatively refractory to PCB99 and PCB153 at levels exceeding those of wildlife and humans, when exposed during adult life. However, the finding of apoptotic Leydig cells merits further investigation.

Guidelines for humane education: alternatives to the use of animals in teaching and training.

There are few areas of animal use that are so emotive as that of their use in education. The physical presence of an animal (whether alive or dead) is a dramatic event for most students, and the effects it has will depend heavily on their previous experience with that species, their moral values and the perceived necessity of the practical. Much of the literature on this subject is highly emotive and based on relatively little data. This paper attempts to clarify the issues raised, presents an overview of the alternatives available with their strengths and weaknesses, and finally offers guidelines for humane education that take into consideration both the practical issues and the feelings of all those involved.

Spontaneous atherosclerosis in the proximal aorta of LPA transgenic mice on a normal diet.

Serum levels of Lp(a) lipoprotein are under genetic control and a high level is a risk factor for atherosclerotic disease. We have examined the aorta of LPA transgenic mice and their non-transgenic litter mates who had all been given a regular, not lipid fortified diet. When sacrificed, the animals had an average age of 66 weeks. Lipid lesions were observed in the aorta of 13 out of 18 LPA transgenic mice and in five out of 21 non-transgenic animals. The difference is statistically significant. We conclude that LPA transgenic mice develop lipid lesions in aorta more frequently than non-transgenic animals, even on a diet with a low fat content. LPA transgenic mice on a normal diet could be a useful animal model for the study of spontaneous human atherosclerosis, its treatment and prevention.