Behl Berthold, Klos Magarethe, Serr Michael, Ebert Ulrich, Janson Bodo, Drescher Karla, Gross Gerhard, Schoemaker Hans
Neuroscience Discovery Research, Abbott, Knollstrasse, D-67008 Ludwigshafen, Germany.
J Neurosci Methods. 2006 Nov 15;158(1):37-49. doi: 10.1016/j.jneumeth.2006.05.011. Epub 2006 Jun 15.
In this study, we describe a new rapid and versatile method to determine the BrdU content of DNA in brain tissues dissected from BrdU-treated rats. Different to already existing BrdU ELISAs the method is suitable for the assessment of BrdU incorporation in ex vivo experiments as it is based on the analysis of tissue extracts instead of immobilized cells. The method comprises the preparation of DNA extracts from dissected tissues, the immobilization of BrdU-containing DNA with an anti-BrdU antibody and quantification of the incorporated BrdU by a peroxidase-conjugated anti-BrdU antibody. Validating the new assay in vitro, we found a clear-cut dependency of the ELISA signal from the time SKNSH neuroblastoma cells had been exposed to BrdU. Parallel studies with existing ELISAs and a parallel immunocytochemical determination of BrdU positive cells revealed comparable results. In vivo experiments showed a virtually linear relationship between the BrdU immunoreactivity in the hippocampus and the time rats have been exposed to BrdU. Repeating the determination of the BrdU content of the same set of tissue samples revealed reproducible relative differences of the ELISA signals. This was true for protocols using purified DNA as well as crude DNA extracts. For the sensitivity and reproducibility of the method heat denaturation of the DNA prior to the analysis in the ELISA was crucial. In rats treated with electroconvulsion the BrdU content of the hippocampus, determined by the new ELISA, was increased to 225% of controls. In a parallel immunohistochemical study, the number of BrdU positive cells was comparably increased to 251% of controls. The assay thus provides a rapid method to detect changes of cell proliferation in dissected brain tissues and other proliferative tissues. With appropriate protocols, the assay may also be used to assess the generation of particular cell types like neurons in neurogenic areas.
在本研究中，我们描述了一种新的快速且通用的方法，用于测定从经5-溴脱氧尿苷（BrdU）处理的大鼠解剖得到的脑组织中DNA的BrdU含量。与现有的BrdU酶联免疫吸附测定（ELISA）不同，该方法适用于评估体外实验中BrdU的掺入情况，因为它基于对组织提取物的分析而非固定化细胞。该方法包括从解剖组织中制备DNA提取物，用抗BrdU抗体固定含BrdU的DNA，并通过过氧化物酶偶联的抗BrdU抗体对掺入的BrdU进行定量。在体外验证新测定法时，我们发现ELISA信号与SKNSH神经母细胞瘤细胞暴露于BrdU的时间有明确的相关性。与现有ELISA的平行研究以及对BrdU阳性细胞的平行免疫细胞化学测定显示了可比的结果。体内实验表明，海马体中的BrdU免疫反应性与大鼠暴露于BrdU的时间之间几乎呈线性关系。重复测定同一组组织样本的BrdU含量显示，ELISA信号的相对差异具有可重复性。对于使用纯化DNA以及粗DNA提取物的方案都是如此。对于该方法的灵敏度和可重复性而言，在ELISA分析之前对DNA进行热变性至关重要。在用惊厥治疗的大鼠中，通过新的ELISA测定，海马体的BrdU含量增加到对照组的225%。在一项平行的免疫组织化学研究中，BrdU阳性细胞的数量相应增加到对照组的251%。因此，该测定法提供了一种快速方法来检测解剖脑组织和其他增殖组织中细胞增殖的变化。通过适当的方案，该测定法还可用于评估神经源性区域中特定细胞类型（如神经元）的生成情况。
