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

Long-term effects of environmentally relevant doses of 2,2',4,4',5,5' hexachlorobiphenyl (PCB153) on neurobehavioural development, health and spontaneous behaviour in maternally exposed mice

Background: Polychlorinated biphenyls (PCBs) are widespread in the environment, human food and breast milk. Seafood is known to contain nutrients beneficial for the normal development and function of the brain, but also contaminants such as PCBs which are neurotoxic. Exposure to non-coplanar PCBs during brain development can disrupt spontaneous behaviour in mice and lead to hyperactive behaviour. Humans are chronically exposed to the highest relative levels of organochlorines in early childhood during brain development, though usually at doses which do not give clinical symptoms of toxicity. This study aimed to elucidate the developmental and behavioural effects of 2,2’,4,4’,5,5’ hexachlorobiphenyl (PCB153) in mice, mimicking human exposure during gestation and lactation. Methods: Environmentally relevant doses of PCB153 were added to the experimental diets. Feed concentrations were approximately 0.5, 6.5, and 1500 μg PCB153/kg feed, representing a realistic and a worst case scenario of frequent consumption of contaminated fish. The study also investigated the effects of maternal nutrition, i.e. a standard rodent diet versus a high inclusion of salmon. Mice pups were examined for physical- and reflex development, sensorimotor function and spontaneous behaviour from five days after birth until weaning. A selection of pups were followed until 16 weeks of age and tested for open field behaviour and the acoustic startle response (ASR) with prepulse inhibition (PPI). Blood thyroid hormones and liver enzymes, blood lipids and PCB153 content in fat were examined at 16 weeks. Statistical analyses modelled the three way interactions of diet, PCB exposure and litter size on behaviour, using generalized linear models (GLM) and linear mixed effect models (LME). The litter was used as a random variable. Non-parametric tests were used for pair wise comparisons of biochemical analyses. Results: Litter size consistently influenced pup development and behaviour. Few lasting PCB153 related changes were observed, but results indicated effects on synchronization of physical development. Perinatal PCB153 exposure appeared to reduce habituation and cause aggression in males, though not statistically significant. Conclusions: Litter size and maternal diet influenced physical development and function more than PCB153 in perinatally exposed mouse pups and supports the developmental importance of maternal care and the social environment

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used <it>Gapdh </it>mRNA and <it>18S </it>ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.</p> <p>Results</p> <p>The abundance of <it>Arbp</it>, <it>Rpl32</it>, <it>Rpl4</it>, <it>Tbp</it>, <it>Polr2a</it>, <it>Hprt1</it>, <it>Pgk1</it>, <it>Ppia </it>and <it>Gapdh </it>mRNA and <it>18S </it>ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.</p> <p>Conclusions</p> <p>The most stable reference genes were <it>Rpl32</it>, <it>Gapdh </it>and <it>Polr2a </it>in mouse post-infarction heart failure, <it>Polr2a</it>, <it>Rpl32 </it>and <it>Tbp </it>in rat post-infarction heart failure and <it>Rpl32 </it>and <it>Pgk1 </it>in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was <it>Rpl32 </it>and <it>Polr2a</it>. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with <it>Rpl4 </it>as was most stable among the tested genes.</p

Cerebral gene expression and neurobehavioural development after perinatal exposure to an environmentally relevant polybrominated diphenylether (BDE47)

Nutrients in seafood are known to be beneficial for brain development. Effects of maternal exposure to 2,2′,4,4′ tetrabromo diphenylether (BDE47) was investigated, alongside the potential ameliorating impact of seafood nutrients, through assessment of neurobehaviour and gene expression in brain and liver. Developing mice were exposed during gestation and lactation via dams dosed through casein- or salmon-based feed, spiked with BDE47. Two concentrations were used: a low level (6 μg/kg feed) representing an environmentally realistic concentration and a high level (1,900 μg/kg feed) representing a BDE47 intake much higher than expected from frequents consumption of contaminated seafood. Experimental groups were similar with respect to reproductive success, growth and physical development. Minor, transient changes in neurobehavioural metrics were observed in groups given the highest dose of BDE47. No significant differences in behaviour or development were seen on postnatal day 18 among maternally exposed offspring. Cerebral gene expression investigated by microarray analyses and validated by RT-qPCR showed low fold changes for all genes, despite dose-dependent accumulation of BDE47 in brain tissue. The gene for glutamate ammonia ligase was upregulated compared to control in the casein-based high BDE47diet, suggesting potential impacts on downstream synaptic transmission. The study supported a previously observed regulation of Igfbp2 in brain with BDE47 exposure. Genes for hepatic metabolic enzymes were not influenced by BDE47. Potential neurotoxic effects and neurobehavioural aberrations after perinatal exposure to high levels of BDE47 were not readily observed in mice pups with the present experimental exposure regimes and methods of analysis

Intake of farmed Atlantic salmon fed soybean oil increases insulin resistance and hepatic lipid accumulation in mice

BACKGROUND: To ensure sustainable aquaculture, fish derived raw materials are replaced by vegetable ingredients. Fatty acid composition and contaminant status of farmed Atlantic salmon (Salmo salar L.) are affected by the use of plant ingredients and a spillover effect on consumers is thus expected. Here we aimed to compare the effects of intake of Atlantic salmon fed fish oil (FO) with intake of Atlantic salmon fed a high proportion of vegetable oils (VOs) on development of insulin resistance and obesity in mice. METHODOLOGY/PRINCIPAL FINDINGS: Atlantic salmon were fed diets where FO was partly (80%) replaced with three different VOs; rapeseed oil (RO), olive oil (OO) or soy bean oil (SO). Fillets from Atlantic salmon were subsequently used to prepare Western diets (WD) for a mouse feeding trial. Partial replacement of FO with VOs reduced the levels of polychlorinated biphenyls (PCB) and dichloro-diphenyl-tricloroethanes (DDT) with more than 50% in salmon fillets, in WDs containing the fillets, and in white adipose tissue from mice consuming the WDs. Replacement with VOs, SO in particular, lowered the n-3 polyunsaturated fatty acid (PUFA) content and increased n-6 PUFA levels in the salmon fillets, in the prepared WDs, and in red blood cells collected from mice consuming the WDs. Replacing FO with VO did not influence obesity development in the mice, but replacement of FO with RO improved glucose tolerance. Compared with WD-FO fed mice, feeding mice WD-SO containing lower PCB and DDT levels but high levels of linoleic acid (LA), exaggerated insulin resistance and increased accumulation of fat in the liver. CONCLUSION/SIGNIFICANCE: Replacement of FO with VOs in aqua feed for farmed salmon had markedly different spillover effects on metabolism in mice. Our results suggest that the content of LA in VOs may be a matter of concern that warrants further investigation

Cerebral gene expression in response to single or combined gestational exposure to methylmercury and selenium through the maternal diet

Controversy remains regarding the safety of consuming certain types of seafood, particularly during pregnancy. While seafood is rich in vital nutrients, it may also be an important source of environmental contaminants such as methylmercury (MeHg). Selenium (Se) is one essential element present in seafood, hypothesised to ameliorate MeHg toxicity. The aim of the present study was to ascertain the impact of Se on MeHg-induced cerebral gene expression in a mammalian model. Microarray analysis was performed on brain tissue from 15-day-old mice that had been exposed to MeHg throughout development via the maternal diet. The results from the microarray analysis were validated using qPCR. The exposure groups included: MeHg alone (2.6 mg kg−1), Se alone (1.3 mg kg−1), and MeHg + Se. MeHg was presented in a cysteinate form, and Se as Se–methionine, one of the elemental species occurring naturally in seafood. Eight genes responded to Se exposure alone, five were specific to MeHg, and 63 were regulated under the concurrent exposure of MeHg and Se. Significantly enriched functional classes relating to the immune system and cell adhesion were identified, highlighting potential ameliorating mechanisms of Se on MeHg toxicity. Key developmental genes, such as Wnt3 and Sparcl1, were also identified as putative ameliorative targets. This study, utilising environmentally realistic forms of toxicants, delivered through the natural route of exposure, in association with the power of transcriptomics, highlights significant novel information regarding putative pathways of selenium and MeHg interaction in the mammalian brain