Expression of nitric oxide synthase isoforms and nitrotyrosine formation after hypoxia-ischemia in the neonatal rat brain.
OBJECTIVE
Production of nitric oxide is thought to play an important role in neuroinflammation. Previously, we have shown that combined inhibition of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) can reduce hypoxia-ischemia-induced brain injury in 12-day-old rats. The aim of this study was to analyze changes in expression of nNOS, iNOS and endothelial NOS (eNOS), and nitrotyrosine (NT) formation in proteins in neonatal rats up to 48 h after cerebral hypoxia-ischemia.


METHODS
Twelve-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia, resulting in unilateral cerebral damage. NOS and nitrotyrosine expression were determined by immunohistochemistry and Western blot analysis at 30 min-48 h after hypoxia-ischemia.


RESULTS
nNOS was increased in both hemispheres from 30 min to 3 h after hypoxia-ischemia. In the contralateral hemisphere, eNOS was decreased 1-3 h after hypoxia-ischemia. In the ipsilateral hemisphere, eNOS was decreased at 0.5 h after hypoxia-ischemia, normalized at 1-3 h and was increased 6-12 h after hypoxia-ischemia. At 24 and 48 h after hypoxia-ischemia, eNOS levels normalized. Surprisingly, iNOS expression did not change from 30 min up to 48 h after hypoxia-ischemia in the ipsi- or contralateral hemisphere. In addition, the regional expression of iNOS in the brain as determined by immunohistochemistry did not change after hypoxia-ischemia. Expression of nitrotyrosine was slightly increased in both hemispheres only at 30 min after hypoxia-ischemia.


CONCLUSIONS
In 12-day-old rat pups, cerebral hypoxia-ischemia induced a transient increase in nNOS, eNOS, and nitrotyrosine in proteins, but no change in iNOS expression up to 48 h after the insult.
van den Tweel
Evelyn R W
ER
Department of Neonatology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.
Nijboer
Cora
C
Kavelaars
Annemieke
A
Heijnen
Cobi J
CJ
Groenendaal
Floris
F
van Bel
Frank
F
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D004195:Disease Models, Animal; D005260:Female; D007839:Functional Laterality; D005786:Gene Expression Regulation / Q000502:physiology*; D020925:Hypoxia-Ischemia, Brain / Q000201:enzymology* / Q000473:pathology; D050112:Imino Pyranoses / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010880:Piperidines / Q000378:metabolism; D051381:Rats; D018709:Statistics, Nonparametric; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D050112:Imino Pyranoses; D010880:Piperidines; C098432:isofagomine; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase