Automated analysis of global ischemia-induced CA1 neuronal death using terminal UTP nick end labeling (TUNEL)

In the brain, DNA fragmentation is associated with apoptotic cell death following ischemic/excitotoxic damage. Fragmented DNA can be detected in situ by labeling the 3'OH termini of the internucleosomal generated fragments with deoxynucleotides, through a process known as terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling, or TUNEL. TUNEL is frequently being used to assess neuronal death following cerebral ischemia in a number of animal models. However, conventional techniques for TUNEL can be time consuming, and are often subjective and thus can lead to inconsistencies among investigators. Moreover, the lack of tools for its quantification and standardization limits the use of this technique in assessing the magnitude of cell death. In the present report, we describe an improved higher throughput technique for TUNEL staining at room temperature on a BioGenex automated stainer, and its subsequent quantitative analysis using NORTHERN ECLIPSE, an imaging analysis program. Its implementation allows us to effectively quantify TUNEL positive cells in the CA1 region of the hippocampus following global forebrain ischemia in rats. We conclude that this general histological technique can be applied to the study of cell death in numerous other experimental models.