RESUMO
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.
