Histology and Infarct Volume Determination
While there are many and in part very different staining protocols for determining and calculating infarct volumes after experimental stroke in rodents, this plethora can ultimately be reduced to a few basic methods, such as histological staining, contras
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roduction One of the first and most important steps when planning and designing a stroke experiment is deciding how to treat the brain tissue of the experimental animals. The wrong decision will cause problems later on, so this step calls for timely and careful considera tion. The researcher’s decision, in turn, depends on having a clear idea of which endpoints will be analyzed. This seemingly trivial aspect is frequently ignored in practice. While determination of the infarct volume is certainly one important issue in experimental stroke research, it is nevertheless only one parameter among several. Analyses of gene expression, protein expression, protein synthesis, Ulrich Dirnagl (ed.), Rodent Models of Stroke, Neuromethods, vol. 47, DOI 10.1007/978-1-60761-750-1_15, © Springer Science+Business Media, LLC 2010
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pH, glucose, lactate, ATP content, regional blood flow, and receptor binding densities are just a few examples of potential complementary investigations which might call for a specific way of tissue pretreatment. Taking all this into consideration, the first step should be to define the endpoints that are to be analyzed. The next step is to check whether it is possible to do all experiments using the same brain tissue or whether different groups are needed for the various investigations. When performing morphological analyses, one has to decide whether or not the brain will be perfused. If perfusion is necessary, two possibilities arise: perfusion with saline to simply wash out brain vessels or perfusion, e.g., with paraformaldehyde (PFA), for additional fixation of the brain tissue. The next decisions concern the tissue workup steps. Tissue can be used natively or it can be prepared, e.g., for cryostat sections, for free-floating vibratome sections or for paraffin embedding. While infarct volume calculation is in principle possible with all of these workup methods, plans to perform additional experiments may influence the decision. For example, if complementary immunohistochemical investigations are planned, the choice may be narrowed down to native cryostat material or PFA-fixed tissue because only they would allow work with certain specific antibodies. If brilliant morphology is of major importance, paraffin-embedded material will provide the best results. For infarct volume determination, one can choose among different staining methods. All procedures have their advantages and disadvantages, so the researcher should know and carefully consider all potential options. Although there is no ideal solution, most options are left open by the use of serial cryostat sections allowing multimodal imaging (1). In the context of infarct volume determination, several terms and definitions are briefly outlined to avoid possible misunderstandings. Infarct is defined as pannecrosis and has to be distinguished from selective neuronal death occurring after milder ischemic events. Depending on the ischemia model used, the size of the infarct may vary significantly, i.e., from large pannecrosis as seen a
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