Evidence for oxidative stress in the developing cerebellum of the rat after chronic mild carbon monoxide exposure (0.0025% in air).
BACKGROUND
The present study was designed to test the hypothesis that chronic very mild prenatal carbon monoxide (CO) exposure (25 parts per million) subverts the normal development of the rat cerebellar cortex. Studies at this chronic low CO exposure over the earliest periods of mammalian development have not been performed to date. Pregnant rats were exposed chronically to CO from gestational day E5 to E20. In the postnatal period, rat pups were grouped as follows: Group A: prenatal exposure to CO only; group B: prenatal exposure to CO then exposed to CO from postnatal day 5 (P5) to P20; group C: postnatal exposure only, from P5 to P20, and group D, controls (air without CO). At P20, immunocytochemical analyses of oxidative stress markers, and structural and functional proteins were assessed in the cerebellar cortex of the four groups. Quantitative real time PCR assays were performed for inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) nitric oxide synthases.


RESULTS
Superoxide dismutase-1 (SOD1), SOD2, and hemeoxygenase-1 (HO-1) immunoreactivity increased in cells of the cerebellar cortex of CO-exposed pups. INOS and nitrotyrosine immunoreactivity also increased in blood vessels and Purkinje cells (PCs) of pups from group-A, B and C. By contrast, nNOS immunoreactivity decreased in PCs from group-B. Endothelial NOS immunoreactivity showed no changes in any CO-exposed group. The mRNA levels for iNOS were significantly up-regulated in the cerebellum of rats from group B; however, mRNA levels for nNOS and eNOS remained relatively unchanged in groups A, B and C. Ferritin-H immunoreactivity increased in group-B. Immunocytochemistry for neurofilaments (structural protein), synapsin-1 (functional protein), and glutamic acid decarboxylase (the enzyme responsible for the synthesis of the inhibitory neurotransmitter GABA), were decreased in groups A and B. Immunoreactivity for two calcium binding proteins, parvalbumin and calbindin, remained unchanged. The immunoreactivity of the astrocytic marker GFAP increased after prenatal exposure.


CONCLUSIONS
We conclude that exogenously supplied CO during the prenatal period promotes oxidative stress as indicated by the up-regulation of SOD-1, SOD-2, HO-1, Ferritin-H, and iNOS with increased nitrotyrosine in the rat cerebella suggesting that deleterious and protective mechanisms were activated. These changes correlate with reductions of proteins important to cerebellar function: pre-synaptic terminals proteins (synapsin-1), proteins for the maintenance of neuronal size, shape and axonal quality (neurofilaments) and protein involved in GABAergic neurotransmission (GAD). Increased GFAP immunoreactivity after prenatal CO-exposure suggests a glial mediated response to the constant presence of CO. There were differential responses to prenatal vs. postnatal CO exposure: Prenatal exposure seems to be more damaging; a feature exemplified by the persistence of markers indicating oxidative stress in pups at P20, following prenatal only CO-exposure. The continuation of this cellular environment up to day 20 after CO exposure suggests the condition is chronic. Postnatal exposure without prenatal exposure shows the least impact, whereas prenatal followed by postnatal exposure exhibits the most pronounced outcome among the groups.
Lopez
Ivan A
IA
Mental Retardation Research Center, Neuroscience Research Building, Room 260C, 635 Charles E Young Drive South, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-7332, USA. ivan@hnsurg.medsch.ucla.edu
Acuna
Dora
D
Beltran-Parrazal
Luis
L
Lopez
Ivan E
IE
Amarnani
Abhimanyu
A
Cortes
Max
M
Edmond
John
J
D000818:Animals; D000831:Animals, Newborn; D001052:Apoferritins / Q000378:metabolism; D002249:Carbon Monoxide Poisoning / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D002531:Cerebellum / Q000196:embryology* / Q000254:growth & development* / Q000473:pathology / Q000503:physiopathology*; D004195:Disease Models, Animal; D004622:Embryo, Mammalian; D005260:Female; D051547:Heme Oxygenase-1 / Q000378:metabolism; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011247:Pregnancy; D011297:Prenatal Exposure Delayed Effects / Q000503:physiopathology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013482:Superoxide Dismutase / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D000099156:Superoxide Dismutase 2
D001052:Apoferritins; D051547:Heme Oxygenase-1; D009419:Nerve Tissue Proteins; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D014443:Tyrosine; D000099156:Superoxide Dismutase 2; C002744:3-nitrotyrosine; C000606292:Sod1 protein, rat