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

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

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

Electrophysiological and arrhythmogenic effects of 5-hydroxytryptamine on human atrial cells are reduced in atrial fibrillation

5-Hydroxytryptamine (5-HT) is proarrhythmic in atrial cells from patients in sinus rhythm (SR) via activation of 5-HT&lt;sub&gt;4&lt;/sub&gt; receptors, but its effects in atrial cells from patients with atrial fibrillation (AF) are unknown. The whole-cell perforated patch-clamp technique was used to record L-type Ca&lt;sup&gt;2+&lt;/sup&gt; current (&lt;i&gt;I&lt;/i&gt;&lt;sub&gt;CaL&lt;/sub&gt;), action potential duration (APD) and arrhythmic activity at 37 °C in enzymatically isolated atrial cells obtained from patients undergoing cardiac surgery, in SR or with chronic AF. In the AF group, 5-HT (10 μM) produced an increase in &lt;i&gt;I&lt;/i&gt;&lt;sub&gt;CaL&lt;/sub&gt; of 115 ± 21% above control (&lt;i&gt;n&lt;/i&gt; = 10 cells, 6 patients) that was significantly smaller than that in the SR group (232 ± 33%; &lt;i&gt;p&lt;/i&gt; 0.05; &lt;i&gt;n&lt;/i&gt; = 27 cells, 12 patients). Subsequent co-application of isoproterenol (1 μM) caused a further increase in &lt;i&gt;I&lt;/i&gt;&lt;sub&gt;CaL&lt;/sub&gt; in the AF group (by 256 ± 94%) that was greater than that in the SR group (22 ± 6%; p &#60; 0.05). The APD at 50% repolarisation (APD&lt;sub&gt;50&lt;/sub&gt;) was prolonged by 14 ± 3 ms by 5-HT in the AF group (&lt;i&gt;n&lt;/i&gt; = 37 cells, 14 patients). This was less than that in the SR group (27 ± 4 ms; &lt;i&gt;p&lt;/i&gt; &#60; 0.05; &lt;i&gt;n&lt;/i&gt; = 58 cells, 24 patients). Arrhythmic activity in response to 5-HT was observed in 22% of cells in the SR group, but none was observed in the AF group (p &#60; 0.05). Atrial fibrillation was associated with reduced effects of 5-HT, but not of isoproterenol, on &lt;i&gt;I&lt;/i&gt;&lt;sub&gt;CaL&lt;/sub&gt; in human atrial cells. This reduced effect on &lt;i&gt;I&lt;/i&gt;&lt;sub&gt;CaL&lt;/sub&gt; was associated with a reduced APD&lt;sub&gt;50&lt;/sub&gt; and arrhythmic activity with 5-HT. Thus, the potentially arrhythmogenic influence of 5-HT may be suppressed in AF-remodelled human atrium

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

Agents increasing cyclic GMP amplify 5-HT4-elicited positive inotropic response in failing rat cardiac ventricle

Activation of 5-HT4 receptors in failing ventricles elicits a cAMP-dependent positive inotropic response which is mainly limited by the cGMP-inhibitable phosphodiesterase (PDE) 3. However, PDE4 plays an additional role which is demasked by PDE3 inhibition. The objective of this study was to evaluate the effect of cGMP generated by particulate and soluble guanylyl cyclase (GC) on the 5-HT4-mediated inotropic response. Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats, exhibiting heart failure 6 weeks after surgery. Contractility was measured in left ventricular preparations. Cyclic GMP was measured by EIA. In ventricular preparations, ANP or BNP displayed no impact on 5-HT4-mediated inotropic response. However, CNP increased the 5-HT4-mediated inotropic response as well as the β1-adrenoceptor (β1-AR)-mediated response to a similar extent as PDE3 inhibition by cilostamide. Pretreatment with cilostamide eliminated the effect of CNP. Inhibition of nitric oxide (NO) synthase and soluble GC by l-NAME and ODQ, respectively, attenuated the 5-HT4-mediated inotropic response, whereas the NO donor Sin-1 increased this response. The effects were absent during PDE3 inhibition, suggesting cGMP-dependent inhibition of PDE3. However, in contrast to the effects on the 5-HT4 response, Sin-1 inhibited whereas l-NAME and ODQ enhanced the β1-AR-mediated inotropic response. cGMP generated both by particulate (NPR-B) and soluble GC increases the 5-HT4-mediated inotropic response in failing hearts, probably through inhibition of PDE3. β1-AR and 5-HT4 receptor signalling are subject to opposite regulatory control by cGMP generated by soluble GC in failing hearts. Thus, cGMP from different sources is functionally compartmented, giving differential regulation of different Gs-coupled receptors

Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells

An Immunomodulating Fatty Acid Analogue Targeting Mitochondria Exerts Anti-Atherosclerotic Effect beyond Plasma Cholesterol-Lowering Activity in apoE(-/-) Mice

Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator-activated receptors (PPARs) and proliferation of mitochondria. This study aimed to penetrate the effect of TTA on the development of atherosclerotic lesions in apolipoprotein (apo)-E-/- mice fed a high-fat diet containing 0.3% TTA for 12 weeks. These mice displayed a significantly less atherosclerotic development vs control. Plasma cholesterol was increased by TTA administration and triacylglycerol (TAG) levels in plasma and liver were decreased by TTA supplementation, the latter, probably due to increased mitochondrial fatty acid oxidation and reduced lipogenesis. TTA administration also changed the fatty acid composition in the heart, and the amount of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was reduced and increased, respectively. The heart mRNA expression of inducible nitric oxidase (NOS)-2 was decreased in TTA-treated mice, whereas the mRNA level of catalase was increased. Finally, reduced plasma levels of inflammatory mediators as IL-1\u3b1, IL-6, IL-17, TNF-\u3b1 and IFN-\u3b3 were detected in TTA-treated mice. These data show that TTA reduces atherosclerosis in apoE -/- mice and modulates risk factors related to atherosclerotic disorders. TTA probably acts at both systemic and vascular levels in a manner independent of changes in plasma cholesterol, and triggers TAG catabolism through improved mitochondrial function

Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue

BACKGROUND: Relative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study was to evaluate and identify optimal reference genes for use in the relative quantification of gene expression in human epicardial fat depots of lean, overweight and obese subjects. METHODOLOGY/PRINCIPAL FINDINGS: Some of the commonly used reference genes including 18S, ACTB, RPL27, HPRT, CYCA, GAPDH, RPLPO, POLR2A and B2M were quantified using real-time PCR analysis. The expression stability of these genes was evaluated using Genorm, Normfinder and Bestkeeper algorithms. In addition, the effect of sample size on the validation process was studied by randomly categorizing subjects in two cohorts of n = 2 and n = 33. CONCLUSIONS/SIGNIFICANCE: CYCA, GAPDH and RPL27 were identified as the most stable genes common to all three algorithms and both sample sizes. Their use as reference gene pairs might contribute to the enhanced robustness of relative quantification in the studies involving the human epicardial adipose tissue