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 necrotic nuclei in tissue sections. However, application of ISEL on human brain requires conservation of DNA integrity during the postmortem delay (PMD) and good accessibility of fragmented DNA after (prolonged) tissue fixation. We therefore investigated ISEL in relation to PMD and fixation in rat and human brain. Application on a unilateral lesion model in perfused rat brain revealed that prolonged post-fixation strongly diminished ISEL results. However, microwave pre-treatment can counteract these masking effects without inducing nonspecific labeling contralaterally. On the other hand, in briefly post-fixed, perfused brain or immersion-fixed rat and human PMD brain, microwave pre-treatment was deleterious and induced strong nonspecific labeling. In young rat brain, PMD did not influence the low numbers of apoptotic nuclei until 24 hr PMD, when massive nuclear labeling occurred. In human cortex, DNA fragmentation patterns were independent of duration of fixation or PMD and were already present from 4.25 hr PMD onwards. Our data suggest that ISEL on human brain represents antemortem DNA damage rather than PMD artifacts. Furthermore, microwave pre-treatment appears beneficial only in particular fixation conditions.
Lucassen
P J
PJ
Graduate School Neurosciences Amsterdam, Netherlands Institute for Brain Research, The Netherlands.
Chung
W C
WC
Vermeulen
J P
JP
Van Lookeren Campagne
M
M
Van Dierendonck
J H
JH
Swaab
D F
DF
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D001921:Brain / Q000166:cytology* / Q000187:drug effects / Q000473:pathology*; D004247:DNA / Q000032:analysis* / Q000187:drug effects / Q000528:radiation effects; D004249:DNA Damage; D005260:Female; D006652:Histological Techniques; D006801:Humans; D008297:Male; D008872:Microwaves; D008875:Middle Aged; D016202:N-Methylaspartate / Q000494:pharmacology; D011180:Postmortem Changes; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D016202:N-Methylaspartate; D004247:DNA