Ethical Use Of Animals Research Articles

Mutation Analysis in Cultured Cells of Transgenic Rodents.

To comply with guiding principles for the ethical use of animals for experimental research, the field of mutation research has witnessed a shift of interest from large-scale in vivo animal experiments to small-sized in vitro studies. Mutation assays in cultured cells of transgenic rodents constitute, in many ways, viable alternatives to in vivo mutagenicity experiments in the corresponding animals. A variety of transgenic rodent cell culture models and mutation detection systems have been developed for mutagenicity testing of carcinogens. Of these, transgenic Big Blue® (Stratagene Corp., La Jolla, CA, USA, acquired by Agilent Technologies Inc., Santa Clara, CA, USA, BioReliance/Sigma-Aldrich Corp., Darmstadt, Germany) mouse embryonic fibroblasts and the λ Select cII Mutation Detection System have been used by many research groups to investigate the mutagenic effects of a wide range of chemical and/or physical carcinogens. Here, we review techniques and principles involved in preparation and culturing of Big Blue® mouse embryonic fibroblasts, treatment in vitro with chemical/physical agent(s) of interest, determination of the cII mutant frequency by the λ Select cII assay and establishment of the mutation spectrum by DNA sequencing. We describe various approaches for data analysis and interpretation of the results. Furthermore, we highlight representative studies in which the Big Blue® mouse cell culture model and the λ Select cII assay have been used for mutagenicity testing of diverse carcinogens. We delineate the advantages of this approach and discuss its limitations, while underscoring auxiliary methods, where applicable.

Abstract A014: Evaluation of the hollow fiber model as a highly predictive in vivo testing system for the selection of xenograft tumor models in cancer drug discovery

Abstract In cancer drug discovery, lead compounds found to possess the most antitumor efficacy in vitro need to be rapidly and economically prioritized for further analysis in conventional xenograft testing. To bridge this gap, Hollingshead et al. (1995) developed the in vivo hollow fiber model (HFM), a prescreen predicting xenograft efficacy. The screen is based on drug- (but not cell-) permeable fibers loaded with tumor cells that are implanted into mice. Following drug treatment, the number of viable cells in the fibers provides information about the drug's potential in vivo antitumor efficacy. Since one animal can receive up to three intraperitoneally and three subcutaneously implanted fibers, one lead can be simultaneously evaluated against up to three cell lines. To evaluate the predictability of the HFM, two target-specific kinase inhibitors were selected: crizotinib and VX-680. Crizotinib is an FDA-approved drug for NSCL that inhibits the kinases ALK, MET, and ROS1, whereas VX-680 inhibits the AURORA kinase family. Both drugs were run in a proliferation assay against a large panel of tumor cell lines to determine the IC50 value. Subsequently, 9 cell lines representing different inhibitor potencies (high, medium, low/none) were chosen for comparison of the proliferation, HFM, and xenograft tumor model results. For crizotinib the cell lines MKN-45, MiaPaca2, and KARPAS-299 were chosen with highly potent inhibition whereas for VX-680 the cell lines HCT-116, Molm-13, and Colo-205 are most potently inhibited in the proliferation assay. Cell lines SKOV-3, LN229, and HT-29 were chosen as noninhibited cell lines for both kinase inhibitors in the in vitro proliferation assay. For each of the 9 cell lines, a subcutaneous xenograft mouse model was established and the effect of both crizotinib and VX680 treatment on tumor growth determined. In parallel, all cell lines were tested in three separate HFMs with 3 cell lines simultaneously. The implanted hollow fibers were analyzed via MTT assay 14 days after therapy initiation. The correlation between the cell-based inhibitory activity and xenograft tumor model results and the correlation between HFM and xenograft tumor model results will be presented. Finally, the implementation of the HFM for the tumor model selection process in cancer drug discovery will be discussed. In case the HFM is a highly predictive in vivo prescreen for target-specific inhibitors, the HFM will reduce costs, time, and most importantly mice consumption according to reduction and refinement, criteria of the three rules (3Rs), guiding principles for more ethical use of animals in drug testing. Citation Format: Annika Sinz, Holger Weber, Cynthia Obodozie. Evaluation of the hollow fiber model as a highly predictive in vivo testing system for the selection of xenograft tumor models in cancer drug discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A014.

Reduction, replacement and ethics of animal use in bio-science research and education

Reduction, replacement and ethics of animal use in bio-science research and education

Considering aspects of the 3Rs principles within experimental animal biology.

The 3Rs - Replacement, Reduction and Refinement - are embedded into the legislation and guidelines governing the ethics of animal use in experiments. Here, we consider the advantages of adopting key aspects of the 3Rs into experimental biology, represented mainly by the fields of animal behaviour, neurobiology, physiology, toxicology and biomechanics. Replacing protected animals with less sentient forms or species, cells, tissues or computer modelling approaches has been broadly successful. However, many studies investigate specific models that exhibit a particular adaptation, or a species that is a target for conservation, such that their replacement is inappropriate. Regardless of the species used, refining procedures to ensure the health and well-being of animals prior to and during experiments is crucial for the integrity of the results and legitimacy of the science. Although the concepts of health and welfare are developed for model organisms, relatively little is known regarding non-traditional species that may be more ecologically relevant. Studies should reduce the number of experimental animals by employing the minimum suitable sample size. This is often calculated using power analyses, which is associated with making statistical inferences based on the P-value, yet P-values often leave scientists on shaky ground. We endorse focusing on effect sizes accompanied by confidence intervals as a more appropriate means of interpreting data; in turn, sample size could be calculated based on effect size precision. Ultimately, the appropriate employment of the 3Rs principles in experimental biology empowers scientists in justifying their research, and results in higher-quality science.

Listening to Horses

The involvement of nonhuman animals in human sport and leisure raises questions about the ethics of animal use (and sometimes abuse) for human pleasure. This article draws on a multispecies ethnography of amateur riding in the United Kingdom to consider some ways in which human participants try to develop attentive relationships with their equine partners. An ethical praxis of paying attention to horses as individual, sentient beings with intrinsic value beyond their relation to human activities can lead to the development of mutually rewarding interspecies relationships and partnerships within sport. However, these relationships always develop within the context of human-centric power relations that position animals as vulnerable subjects, placing moral responsibility on humans to safeguard animal interests in human sport and leisure.

Use of Optical Imaging Technology in the Validation of a New, Rapid, Cost-Effective Drug Screen as Part of a Tiered In Vivo Screening Paradigm for Development of Drugs To Treat Cutaneous Leishmaniasis.

ABSTRACTIn any drug discovery and development effort, a reduction in the time of the lead optimization cycle is critical to decrease the time to license and reduce costs. In addition, ethical guidelines call for the more ethical use of animals to minimize the number of animals used and decrease their suffering. Therefore, any effort to develop drugs to treat cutaneous leishmaniasis requires multiple tiers of in vivo testing that start with higher-throughput efficacy assessments and progress to lower-throughput models with the most clinical relevance. Here, we describe the validation of a high-throughput, first-tier, noninvasive model of lesion suppression that uses an in vivo optical imaging technology for the initial screening of compounds. A strong correlation between luciferase activity and the parasite load at up to 18 days postinfection was found. This correlation allows the direct assessment of the effects of drug treatment on parasite burden. We demonstrate that there is a strong correlation between drug efficacy measured on day 18 postinfection and the suppression of lesion size by day 60 postinfection, which allows us to reach an accurate conclusion on drug efficacy in only 18 days. Compounds demonstrating a significant reduction in the bioluminescence signal compared to that in control animals can be tested in lower-throughput, more definitive tests of lesion cure in BALB/c mice and Golden Syrian hamsters (GSH) using Old World and New World parasites.

Inflammation and immunogenicity limit the utility of the rabbit as a nonclinical species for ocular biologic therapeutics

The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species. Such studies are often confounded by the development of anti-drug antibodies and severe ocular inflammation, the latter requiring studies to be terminated prematurely for animal welfare reasons. Notably, these confounding factors preclude the interpretation of safety. Nonclinical toxicology programs should be designed with consideration of ethical animal use and 3Rs principles (Replacement, Reduction and Refinement). The experience of several pharmaceutical sponsors, demonstrating that toxicology studies of ocular (intravitreal and topical ocular) biologic drug candidates in the rabbit are of limited interpretive value, calls into question the utility of such studies in this species and indicates that such studies should not be conducted.

A novel dissolution method for evaluation of polysaccharide based colon specific delivery systems: A suitable alternative to animal sacrifice

The most extensively used test for predicting in-vivo release kinetics of a drug from its orally administered dosage forms is dissolution testing. For polysaccharide based, colon targeted oral delivery systems, the entire path of the gut traversed by the dosage form needs to be simulated for assessing its in-vivo dissolution pattern. This includes the dissolution testing sequentially in simulated gastric fluid (SGF), simulated intestinal fluid (SIF) and simulated colonic fluid (SCF). For SGF and SIF, simple and standardized composition is well-known. However, preparation of SCF requires addition of either the colonic contents of rodents or human faecal slurry. A method is proposed, wherein a mixture of five probiotics cultured in the presence of a prebiotic under anaerobic conditions is able to surrogate the colonic fluid. Release profiles of drug from colon targeted delivery systems in this medium were studied and compared to those generated in the conventionally used media containing rodent caecal contents and human faecal slurry. The results from the three studies were found to be quite similar. These findings suggest that the proposed medium may prove to be useful not only as a biorelevant and discriminatory method but may also help in achieving the 3Rs objective regarding the ethical use of animals.

Philosophy and ethics of animal use and consumption: from Pythagoras to Bentham.

Abstract The human population is projected to rise to 9.15 billion people by 2050, resulting in a projected increase in meat consumption. However, at the same time, the dominant position of meat as the centre of meals is on the decline, resulting in an increasing number of vegetarians and flexitarians in some Western countries. There are several motivations for this trend, including religious, health, moral and environmental considerations. Many of the arguments that are very alive today in the debate follow from those proposed by ancient philosophers and scientists. The aim of this review is to review the ethics of animal use and consumption from the old Greek philosophers (Pythagoras, Heraclitus, Alcmaeon, Anaxagoras, Socrates, Plato, Aristotle, Diogenes, Theophrastus, the Stoics, the Pyrrhonian Skeptics, Plutarch and the Phoenician philosopher Porphyry), the early Christians and the Middle Ages, and the Renaissance, to the seventeenth and eighteenth century (Evelyn, Tryon, Descartes, Malebranche, De La Mettrie, Kant, Spinoza and Bentham).

Similar but not the same: the teaching of veterinary and medical ethics

THE inclusion of veterinary ethics as a discrete and recognisable subject in veterinary curricula has been a creeping progression rather than a huge leap. In comparison to medical ethics, the...

Evaluation of Regional Adiposity in the Neck Area of Andalusian Horses

Reasons for performing studyAdipose tissue deposited along the crest of the neck has been associated with altered metabolic states and with an increased risk of laminitis in equids. Thickness of the neck is a phenotipic characteristic in Andalusians; however, there are no studies evaluating this condition in this breed.ObjectivesTo evaluate morphometric and ultrasonographic measurements in the neck area for assessment of neck adiposity and its associations with adiposity scores and biochemical variables.Study designA sample of 115 Andalusian horses (46 barren mares and 69 stallions, 2–15 years old) was evaluated in a cross‐sectional study.MethodsCresty neck score (CNS) was determined. Two groups were created: cresty neck (CNS ≥ 3) and noncresty neck (CNS<3) horses. Neck circumference (NC) (cm) and ultrasonography (US) of subcutaneous fat (mm) at 25% (NC0.25–US0.25), 50% (NC0.50–US0.50) and 75% (NC0.75–US0.75) of neck length were measured. Middle neck circumference (NC), neck length and height at the withers were taken to calculate neck ratios (NC0.50:height, NC0.50:neck length). Plasma was analysed for insulin and leptin. Hyperinsulinemia was defined as insulin ≥20 μiu/ml. Correlations using Pearson and Spearman coefficients were evaluated. Mean comparisons were carried out with Student's t or Mann‐Whitney tests.ResultsCresty neck condition was present in 73.9% (n = 85) of the horses and 2.6% (n = 3) were hyperinsulinaemic. Cresty neck horses had significantly higher plasma leptin, morphometric and ultrasonographic measurements. NC0.50:height was the most correlated parameter with CNS. Insulin was correlated with leptin levels and all morphometric measurements. Insulin and leptin were correlated only with US0.75.ConclusionsAndalusian horses’ neck score, despite their thick necks in the majority of individuals, does not have a good correlation with plasma insulin and leptin levels. Since in Andalusian horses, neck scores may be overestimating obesity, further studies on ultrasonographic fat measurements are needed.Ethical animal research: The university committee for the ethical use of animals approved all procedures. Owners gave informed consent for their horses’ inclusion in the study. Sources of funding: None. Competing interests: None.

Ethical animal use in education and training: from clinical rotations to ethically sourced cadavers.

The InterNICHE Policy defines the concept of ‘ethically sourced’ material to encourage replacement and enhance the acquisition of knowledge, skills and attitudes

What's Special about the Ethical Challenges of Studying Disorders with Altered Brain Activity?

Where there is no viable alternative, studies of neuronal activity are conducted on animals. The use of animals, particularly for invasive studies of the brain, raises a number of ethical issues. Practical or normative ethics are enforced by legislation, in relation to the dominant welfare guidelines developed in the United Kingdom and elsewhere. Guidelines have typically been devised to cover all areas of biomedical research using animals in general, and thus lack any specific focus on neuroscience studies at the level of the ethics, although details of the specific welfare recommendations are different for invasive studies of the brain. Ethically, there is no necessary distinction between neuroscience and other biomedical research in that the brain is a final common path for suffering, irrespective of whether this involves any direct experience of pain. One exception arises in the case of in vitro studies, which are normally considered as an acceptable replacement for in vivo studies. However, to the extent sentience is possible, maintaining central nervous system tissue outside the body naturally raises ethical questions. Perhaps the most intractable challenge to the ethical use of animals in order to model neuronal disorder is presented by the logical impasse in the argument that the animal is similar enough to justify the validity of the experimental model, but sufficiently different in sentience and capacity for suffering, for the necessary experimental procedures to be permissible.

Considerations Regarding Appropriate Sample Size for Conducting Ferret Transmission Experiments

Evaluation of: Nishiura H, Yen H-L, Cowling BJ. Sample size considerations for one-to-one animal transmission studies of the influenza A viruses. PLoS ONE 8(1), e55358 (2013). There is an urgent need to model in a laboratory setting the capacity of wild-type influenza viruses to transmit between mammals, to determine the molecular determinants and identify biological properties that confer influenza virus transmissibility, and to explore both pharmaceutical and nonpharmaceutical methods to inhibit virus transmission. Owing to its close physiologic match to humans, researchers typically utilize the ferret to measure influenza virus transmissibility. Nishiura et al. highlight the dilemma facing researchers utilizing the ferret transmission model: how to provide high-quality data to guide public health efforts, while ensuring the ethical use of animals in limited-size, individual, one-to-one transmission experiments. However, the responsible interpretation of data generated using this model can overcome this potential limitation. A closer examination of previously published studies utilizing this model as it is currently employed reveals that the 'sample size' of these studies is not always as small as it may appear.

Should We Legalise Recreational Drugs? An Ethical Question for Animals and Humans

Recreational drugs have been with us since time immemorial—opium was probably cultured by prehistoric people in the 6 millennium BC and it has been suggested that some otherwise unexplainable fantastical cave drawings reflect recreational drug use [1]. Much more recently, Sir Arthur Conan Doyle stimulated his imagination with cocaine and perhaps opium [2] which likely led to his opium-addicted alter-ego Sherlock Holmes’ exploits; Pete Townshend, of The Who, was probably high on lysergic acid diethylamide (LSD), cocaine or cannabinoids when he composed many of his most celebrated works [3]; and sufferers of debilitating and painful diseases, like multiple sclerosis, find solace in an illegal smoke of cannabis [4] in some countries, whereas they might be able to use cannabis legally in other jurisdictions (e.g. California, USA [5]). But, perhaps more importantly, millions of young people throughout the world party on Saturday night under the influence of a new generation of psychoactive drugs including, synthetic cannabinoids (e.g. JHW-018), BZP and methamphetamine. The question is should we legalise the recreational use of these drugs? Legalising them would mean that their safety would be assessed and the cost of policing and prosecuting illicit drug use would be cut. The Netherlands was the first country to legalise a recreational drug, cannabis, with an arguably successful outcome. But, should we go further? Should such drugs and their synthetic derivatives become legal products marketed by a new pharmaceuticals industry? The New Zealand government has decided to legalise psychoactive recreational drugs and has embarked upon the process of setting the requirements for safety evaluation. There is significant controversy about whether it is ethical to use animals to test drugs with an, at best, dubious benefit. This debate is important because it addresses the question when is the use of animals in drug testing ethically acceptable? Indeed, it was conceivable that this debate might derail the New Zealand government’s Psychoactive Substances Bill (2013) [6], and, as a result, the Parliamentary Select Committee has refused to consider submissions on the ethics of animal use in testing recreational drugs. The rationale behind this decision is that ethics committees will decide whether the use of animals to test recreational drugs is appropriate or not. But it is likely that recreational drug manufacturers will hail from parts of the world where ethics are far from most people’s minds – especially when there a significant financial benefit that depends on animal use. Toxicologists have been through a painful and arduous process (mainly in the 1980s) to understand the ethics of animal use in their work. It took significant action by extremist antivivisectionists and reasonable protesters alike to precipitate a rethink of animal use. Now most toxicologists take the use of animals in their work very seriously and will not use animals unless the outcome of their work is important. Defining ‘important’ is difficult, but in the context of drug safety evaluation this would be that the resulting drug has a benefit that balances the sacrifice of animals as part of its development process.

Animal models of chronic kidney disease: useful but not perfect

Animal models of chronic kidney disease (CKD) approximate the human condition and are keys to understanding its pathogenesis and to developing rational treatment strategies. The ethical use of animals requires a detailed understanding of the strengths and limitations of each species and the disease model, and the way in which findings can be translated from animals to humans. While not perfect, the careful use of animal experiments offers the opportunity to examine individual mechanisms in an accelerated time frame.

Standards for Wildlife Research: Taxon-Specific Guidelines versus US Public Health Service Policy

The Guide for the Care and Use of Laboratory Animals (NRC 2011) serves as the principal reference for the oversight of most vertebrate use in research and teaching in the United States. The Guide was developed as a reference for biomedical research. Beyond guiding ethical principles, the Guide contains little information useful for the oversight of research involving wild taxa. To fill this breach, and at the behest of the National Science Foundation, taxon-specific societies in the United States developed independent guidelines that held to the principles of ethical use of animals in research and that were specific to wildlife. Recognition of these taxon-specific guidelines by federal grantmaking agencies and the animal welfare community as appropriate standards for wildlife research will facilitate the required oversight of research involving wild taxa and the ethical use of wild animals in research and teaching.

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Chronic Opioid Potentiates Presynaptic but Impairs Postsynaptic N-Methyl-d-aspartic Acid Receptor Activity in Spinal Cords: IMPLICATIONS FOR OPIOID HYPERALGESIA AND TOLERANCE

Casein Kinase 2-mediated Synaptic GluN2A Up-regulation Increases N-Methyl-d-aspartate Receptor Activity and Excitability of Hypothalamic Neurons in Hypertension

Increased glutamatergic input, particularly N-methyl-D-aspartate receptor (NMDAR) activity, in the paraventricular nucleus (PVN) of the hypothalamus is closely associated with high sympathetic outflow in essential hypertension. The molecular mechanisms underlying augmented NMDAR activity in hypertension are unclear. GluN2 subunit composition at the synaptic site critically determines NMDAR functional properties. Here, we found that evoked NMDAR-excitatory postsynaptic currents (EPSCs) of retrogradely labeled spinally projecting PVN neurons displayed a larger amplitude and shorter decay time in spontaneously hypertensive rats (SHRs) than in Wistar-Kyoto (WKY) rats. Blocking GluN2B caused a smaller decrease in NMDAR-EPSCs of PVN neurons in SHRs than in WKY rats. In contrast, GluN2A blockade resulted in a larger reduction in evoked NMDAR-EPSCs and puff NMDA-elicited currents of PVN neurons in SHRs than in WKY rats. Blocking presynaptic GluN2A, but not GluN2B, significantly reduced the frequency of miniature EPSCs and the firing activity of PVN neurons in SHRs. The mRNA and total protein levels of GluN2A and GluN2B in the PVN were greater in SHRs than in WKY rats. Furthermore, the GluN2B Ser(1480) phosphorylation level and the synaptosomal GluN2A protein level in the PVN were significantly higher in SHRs than in WKY rats. Inhibition of protein kinase CK2 normalized the GluN2B Ser(1480) phosphorylation level and the contribution of GluN2A to NMDAR-EPSCs and miniature EPSCs of PVN neurons in SHRs. Collectively, our findings suggest that CK2-mediated GluN2B phosphorylation contributes to increased synaptic GluN2A, which potentiates pre- and postsynaptic NMDAR activity and the excitability of PVN presympathetic neurons in hypertension.

Wildlife management and the debate on the ethics of animal use. I. Decisions within a State wildlife agency.

Some actions necessary to conserve wildlife sit uncomfortably with those who are concerned about the ethics of animal use. The statutory framework for protecting wildlife is outlined, and examples of the range of issues faced in a State wildlife management agency are discussed, including city wildlife, invasive species, hunting, keeping native animals, threatened species recovery and preparing for climate change. To maintain public support, government wildlife managers need to engage with the different views in society of how we should treat animals. Palmer (2010), a philosopher, identified three zones — wild, contact, and dependent — where humans and animals interact, each with a different ethical context and requiring a different response from people. Geography can determine attitude and destiny, particularly when an animal is foreign to a place, such as rabbits and foxes in Australia. The concept of native animals as pests and/or commercially valuable species has a complicated history, with shooting and commercial hunting reflecting the first half of the European history of wildlife management in Australia. No one word defines our optimal relationship to animals, be it minding, looking, liberation, protection or management, and this range of words identifies the scale of the test facing wildlife managers tasked with making decisions about wildlife. Sutherland et al. (2009) identified 100 questions of importance to the conservation of global biological diversity. I would expand this to 101, to encourage the active engagement of wildlife managers and conservation biologists in the debate on the ethics of animal use.