Complexities, Confounders, and Challenges in Experimental Stroke Research: A Checklist for Researchers and Reviewers
The quest for internal and external validity in experimental stroke research is fraught with pitfalls and confounders. This article, written as a checklist from the perspective of an editor and reviewer of articles on rodent stroke models and an active be
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1. Introduction Modeling disease in animals is highly complex. Many confounders challenge the internal and external validity of this type of research. Modeling stroke is particularly challenging with respect to bias and confounders: It involves damage to the most complex organ in the known universe and produces a plethora of secondary changes in peripheral metabolism as well as in the endocrine, cardiovascular, and immune systems which occur after stroke-induced failures in the brain’s homeostatic control function. As an editor and reviewer of articles in the cardiovascular field and as a stroke researcher, I am constantly faced with these complexities and with
Ulrich Dirnagl (ed.), Rodent Models of Stroke, Neuromethods, vol. 47, DOI 10.1007/978-1-60761-750-1_19, © Springer Science+Business Media, LLC 2010
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the various strategies (and sometimes failures) for overcoming them and maintaining quality. In this chapter, I have compiled the most common and relevant pitfalls and quality issues in experimental stroke research in the form of a checklist. It is meant to guide the experimentalist in planning stroke experiments and the reviewer of experimental stroke research in assessing such research. Many of the issues introduced here are covered in greater detail in the chapters which follow.
2. The Checklist Proper controls for experiments with genetically modified animals?
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The potential for manipulating gene expression in vivo by deleting (knockout) or inserting genes (knock-in, transgenics) has revolutionized biological research and provided highly relevant insights into mechanisms of disease. Genetically manipulated animals are among the mainstays of modern cerebral ischemia research. However, their use brings with it several highly relevant confounders which need to be taken into account when planning an experimental stroke study. In particular, we need to be aware of genetic background, flanking genes, and insertion-site effects. The flanking gene problem (1) is of particular relevance when embryonic stem (ES) cells for the generation of the knockout mouse have been derived from substrains of the 129 inbred mice, whose germ lines will transmit not only the induced null mutation, but also the 129 genetic background. Mating these mice with another inbred strain, very often the C57BL/6, results in an F2 generation that segregates not only for the induced null mutation and its wild-type allele, but also for any other alleles at loci where the parental strains differ (2). The C57BL/6 and 129 mouse strains are highly popular, not only in the genetic engineering of mice, but also in experimental stroke research. Solutions to the flanking gene problem include backcrossing and outcrossing strategies that produce congenic or coisogenic lines (1). Due to gross differences in the vascularization of the brain (3–5) and intrinsic factors of which we understand very little (6), C57BL/6 and 129 mouse strains differ greatly in their susceptibility to cerebral ischemia. Therefore, the flanking gene problem i
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