In situ detection of apoptotic nuclei in the substantia nigra compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice using terminal deoxynucleotidyl transferase labelling and acridine orange staining
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
The neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was used to generate
a dose-dependent cell death of dopaminergic nigral neurons in the C57B1 mouse. Mice
were injected with a total cumulative dose of 150 mg/kg of MPTP delivered over five
days and killed at different time points both during and after the toxin injections.
Two independent histological methods were used to determine whether the dopaminergic
nigral neurons died via an apoptotic mechanism. In situ end-labelling with terminal
deoxynucleotidyl transferase was used on paraformaldehyde-fixed, serial, frozen sections
to identity cells with double-stranded DNA breaks. Apoptotic cell death was found
to be initiated within 72 h of the first injection of the neurotoxin and peaked 24
h after the final MPTP injection. The metachromatic fluorochrome, Acridine Orange,
was used on alternate sections to provide structural confirmation of the nuclear chromatin
"clumping" considered to be representative of apoptosis. Confocal laser imaging combined
with deconvolution techniques was used to resolve the fluorescent signal emitted by
the in situ Acridine Orange-DNA complexes. The number of Acridine Orange-stained nuclei
demonstrating chromatin clumping was identical to that of the positive in situ end-labelled
nuclei observed over a 25 day period. Based upon these two independent methods of
assessing apoptosis in situ, we conclude that a 150 mg/kg dose of MPTP can elicit
apoptotic cell death in nigral dopaminergic neurons of the C57B1 mouse.