Motor and cognitive deficits in the heterozygous leaner mouse, a Cav2.1 voltage-gated Ca2+ channel mutant

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neurobiolaging.The leaner mutation in mice affects the Ca(v)2.1 voltage-gated calcium channel alpha(1A)-subunit gene (Cacna1a), causing a reduction in calcium currents predominantly in Purkinje cells. This reduction in calcium currents causes severe progressive cerebellar ataxia, beginning around postnatal day 10, in homozygous leaner mice (tg(la)/tg(la)), while their heterozygous littermates (tg(la)/+) present no obvious behavioral deficits. In humans, heterozygous mutations in the Cacna1a orthologous gene produce a broad range of neurological manifestations. To evaluate the phenotypic status of the tg(la)/+ animals, we assessed motor performance and cognition, at different ages, in these mutant mice. We were able to observe age-dependent impairment in motor and cognitive tasks; balance and motor learning deficits were found in demanding tasks on the rotarod and on the hanging wire test, while spatial learning and memory impairment was observed in the Morris water maze. Progressive dysfunction in escape reflexes, indicative of neurological impairment, was also present in tg(la)/+ animals. Although not presenting major motor alterations, tg(la)/+ mice show age-dependent motor and cognitive deficits.We would like to thank Carolina Lemos for her help with statistical analysis and Victor Mendes for image technical assistance. This work was supported by research grants POCTI/MGI/34517/00, POCTI/NSE/45352/2002 and POCI/SAU-MMO/56387/2004, FCT (Fundação para a Ciência e Tecnologia) and co-funded by FEDER. I.A. is recipient of a scholarship from FCT, Portugal

Advancing the 3Rs: innovation, implementation, ethics and society

The 3Rs principle of replacing, reducing and refining the use of animals in science has been gaining widespread support in the international research community and appears in transnational legislation such as the European Directive 2010/63/EU, a number of national legislative frameworks like in Switzerland and the UK, and other rules and guidance in place in countries around the world. At the same time, progress in technical and biomedical research, along with the changing status of animals in many societies, challenges the view of the 3Rs principle as a sufficient and effective approach to the moral challenges set by animal use in research. Given this growing awareness of our moral responsibilities to animals, the aim of this paper is to address the question: Can the 3Rs, as a policy instrument for science and research, still guide the morally acceptable use of animals for scientific purposes, and if so, how? The fact that the increased availability of alternatives to animal models has not correlated inversely with a decrease in the number of animals used in research has led to public and political calls for more radical action. However, a focus on the simple measure of total animal numbers distracts from the need for a more nuanced understanding of how the 3Rs principle can have a genuine influence as a guiding instrument in research and testing. Hence, we focus on three core dimensions of the 3Rs in contemporary research: (1) What scientific innovations are needed to advance the goals of the 3Rs? (2) What can be done to facilitate the implementation of existing and new 3R methods? (3) Do the 3Rs still offer an adequate ethical framework given the increasing social awareness of animal needs and human moral responsibilities? By answering these questions, we will identify core perspectives in the debate over the advancement of the 3Rs

Taking ethics into account in farm animal breeding:what can the breeding companies achieve?

Animal welfare and the ethical issues it raises have been discussed intensively for a couple of decades. The emphasis has been on the direct effects of housing and husbandry, but more attention is now being given to problems originating in selective breeding. European attempts to adjust animal welfare legislation to deal with these problems have been largely unsuccessful, but the fact that selective breeding can introduce welfare problems continues to place an ethical responsibility on the animal breeding industry. Since breeding decisions are made centrally and, increasingly, internationally, strategic change is only likely to occur if it is embedded in an international agreement of some kind. The aim of this paper is to describe the key ethical issues facing animal breeding and assess the suggestion that the breeding industry itself can deal with ethical issues by means of an ethical code. Results from recent projects involving commercial breeding enterprises are presented

Harm–benefit analysis – what is the added value?:A review of alternative strategies for weighing harms and benefits as part of the assessment of animal research

Animal experiments are widely required to comply with the 3Rs, to minimise harm to the animals and to serve certain purposes in order to be ethically acceptable. Recently, however, there has been a drift towards adding a so-called harm-benefit analysis as an additional requirement in assessing experiments. According to this, an experiment should only be allowed if there is a positive balance when the expected harm is weighed against the expected benefits. This paper aims to assess the added value of this requirement. Two models, the discourse model and the metric model, are presented. According to the former, the weighing of harms and benefits must be conducted by a committee in which different stakeholders engage in a dialogue. Research into how this works in practice, however, shows that in the absence of an explicit and clearly defined methodology, there are issues about transparency, consistency and fairness. According to the metric model, on the other hand, several dimensions of harms and benefits are defined beforehand and integrated in an explicit weighing scheme. This model, however, has the problem that it makes no real room for ethical deliberation of the sort committees undertake, and it has therefore been criticised for being too technocratic. Also, it is unclear who is to be held accountable for built-in ethical assumptions. Ultimately, we argue that the two models are not mutually exclusive and may be combined to make the most of their advantages while reducing the disadvantages of how harm-benefit analysis in typically undertaken

Retrospective harm benefit analysis of pre-clinical animal research for six treatment interventions

The harm benefit analysis (HBA) is the cornerstone of animal research regulation and is considered to be a key ethical safeguard for animals. The HBA involves weighing the anticipated benefits of animal research against its predicted harms to animals but there are doubts about how objective and accountable this process is.i. To explore the harms to animals involved in pre-clinical animal studies and to assess these against the benefits for humans accruing from these studies; ii. To test the feasibility of conducting this type of retrospective HBA.Data on harms were systematically extracted from a sample of pre-clinical animal studies whose clinical relevance had already been investigated by comparing systematic reviews of the animal studies with systematic reviews of human studies for the same interventions (antifibrinolytics for haemorrhage, bisphosphonates for osteoporosis, corticosteroids for brain injury, Tirilazad for stroke, antenatal corticosteroids for neonatal respiratory distress and thrombolytics for stroke). Clinical relevance was also explored in terms of current clinical practice. Harms were categorised for severity using an expert panel. The quality of the research and its impact were considered. Bateson's Cube was used to conduct the HBA.The most common assessment of animal harms by the expert panel was 'severe'. Reported use of analgesia was rare and some animals (including most neonates) endured significant procedures with no, or only light, anaesthesia reported. Some animals suffered iatrogenic harms. Many were kept alive for long periods post-experimentally but only 1% of studies reported post-operative care. A third of studies reported that some animals died prior to endpoints. All the studies were of poor quality. Having weighed the actual harms to animals against the actual clinical benefits accruing from these studies, and taking into account the quality of the research and its impact, less than 7% of the studies were permissible according to Bateson's Cube: only the moderate bisphosphonate studies appeared to minimise harms to animals whilst being associated with benefit for humans.This is the first time the accountability of the HBA has been systematically explored across a range of pre-clinical animal studies. The regulatory systems in place when these studies were conducted failed to safeguard animals from severe suffering or to ensure that only beneficial, scientifically rigorous research was conducted. Our findings indicate a pressing need to: i. review regulations, particularly those that permit animals to suffer severe harms; ii. reform the processes of prospectively assessing pre-clinical animal studies to make them fit for purpose; and iii. systematically evaluate the benefits of pre-clinical animal research to permit a more realistic assessment of its likely future benefits

Genome-wide association study identifies a variant in HDAC9 associated with large vessel ischemic stroke

Genetic factors have been implicated in stroke risk but few replicated associations have been reported. We conducted a genome-wide association study (GWAS) in ischemic stroke and its subtypes in 3,548 cases and 5,972 controls, all of European ancestry. Replication of potential signals was performed in 5,859 cases and 6,281 controls. We replicated reported associations between variants close to PITX2 and ZFHX3 with cardioembolic stroke, and a 9p21 locus with large vessel stroke. We identified a novel association for a SNP within the histone deacetylase 9(HDAC9) gene on chromosome 7p21.1 which was associated with large vessel stroke including additional replication in a further 735 cases and 28583 controls (rs11984041, combined P = 1.87×10−11, OR=1.42 (95% CI) 1.28-1.57). All four loci exhibit evidence for heterogeneity of effect across the stroke subtypes, with some, and possibly all, affecting risk for only one subtype. This suggests differing genetic architectures for different stroke subtypes