Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to postmortem delay and fixation of rat and human brain.

In situ end-labeling (ISEL) identifies DNA fragmentation in apoptotic or necroticnuclei in tissue sections. However, application of ISEL on human brain requiresconservation of DNA integrity during the postmortem delay (PMD) and goodaccessibility of fragmented DNA after (prolonged) tissue fixation. We thereforeinvestigated ISEL in relation to PMD and fixation in rat and human brain. Applicationon a unilateral lesion model in perfused rat brain revealed that prolongedpost-fixation strongly diminished ISEL results. However, microwave pre-treatment cancounteract these masking effects without inducing nonspecific labelingcontralaterally. On the other hand, in briefly post-fixed, perfused brain orimmersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious andinduced strong nonspecific labeling. In young rat brain, PMD did not influence thelow numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labelingoccurred. In human cortex, DNA fragmentation patterns were independent of duration offixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggestthat ISEL on human brain represents antemortem DNA damage rather than PMD artifacts.Furthermore, microwave pre-treatment appears beneficial only in particular fixationconditions.