Nitrosylation precedes caspase-3 activation and translocation of apoptosis-inducing factor in neonatal rat cerebral hypoxia-ischaemia.
Excessive nitric oxide (NO) production after cerebral hypoxia-ischaemia (HI) may induce cellular injury in various ways, including reaction with superoxide to form the highly reactive peroxynitrite. We characterized the spatial and temporal formation of peroxynitrite through immunohistochemical detection of nitrosylated proteins. Nitrotyrosine immunoreactivity peaked around 3 h post-HI and was detected in areas of injury, as judged by the loss of microtubule-associated protein-2 (MAP-2) staining, in neurones, glia and endothelial cells. Nitrotyrosine staining co-localized with three other cellular markers of injury, active caspase-3, nuclear translocation of apoptosis-inducing factor (AIF) and an oligonucleotide hairpin probe detecting specific DNA strand breaks. The number of nitrotyrosine-positive cells at early time points outnumbered the cells positive for the other three markers of injury, indicating that nitrosylation preceded caspase-3 activation. Pharmacological inhibition of neuronal and inducible nitric oxide synthase (nNOS and iNOS) using 2-iminobiotin, which has been demonstrated earlier to be neuroprotective, significantly reduced nitrotyrosine formation and caspase-3 activation, but not nuclear translocation of AIF, in cortex and striatum of the ipsilatral hemisphere. In summary, nitrotyrosine is an early marker of cellular injury and inhibition of nNOS and iNOS is a promising strategy for neuroprotection after perinatal HI.
Zhu
Changlian
C
Department of Physiology, Göteborg University, Göteborg, Sweden. changlian.zhu@fysiologi.gu.se
Wang
Xiaoyang
X
Qiu
Lin
L
Peeters-Scholte
Cacha
C
Hagberg
Henrik
H
Blomgren
Klas
K
D000818:Animals; D000831:Animals, Newborn; D051033:Apoptosis Inducing Factor; D015415:Biomarkers / Q000032:analysis; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002452:Cell Count; D004249:DNA Damage; D004195:Disease Models, Animal; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005420:Flavoproteins / Q000378:metabolism*; D020925:Hypoxia-Ischemia, Brain / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
C494831:Aifm1 protein, rat; D051033:Apoptosis Inducing Factor; D015415:Biomarkers; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D008565:Membrane Proteins; C002744:3-nitrotyrosine; D014443:Tyrosine; D001710:Biotin; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; C496322:Nos1 protein, rat; C496318:Nos2 protein, rat; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; C028072:2-iminobiotin