Effects of oxygen and glucose deprivation on the expression and distribution of neuronal and inducible nitric oxide synthases and on protein nitration in rat cerebral cortex.
Changes in the nitric oxide (NO) system of the rat cerebral cortex were investigated by immunohistochemistry, immunoblotting, NO synthase (NOS) activity assay, and magnetic resonance imaging (MRI) in an experimental model of global cerebral ischemia and reperfusion. Brains were perfused transcardially with an oxygenated plasma substitute and subjected to 30 minutes of oxygen and glucose deprivation, followed by reperfusion for up to 12 hours with oxygenated medium containing glucose. A sham group was perfused without oxygen or glucose deprivation, and a further group was treated with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) before and during perfusion. Global ischemia led to cerebrocortical injury as shown by diffusion MRI. This was accompanied by increasing morphologic changes in the large type I interneurons expressing neuronal NOS (nNOS) and the appearance of nNOS immunoreactivity in small type II neurons. The nNOS-immunoreactive band and calcium-dependent NOS activity showed an initial increase, followed by a fall after 6 hours of reperfusion. Inducible NOS immunoreactivity appeared in neurons, especially pyramidal cells of layers IV-V, after 4 hours of reperfusion, with corresponding changes on immunoblotting and in calcium-independent NOS activity. Immunoreactive protein nitrotyrosine, present in the nuclear area of neurons in nonperfused controls and sham-perfused animals, showed changes in intensity and distribution, appearing in the neuronal processes during the reperfusion period. Prior and concurrent L-NAME administration blocked the changes on diffusion MRI and attenuated the morphologic changes, suggesting that NO and consequent peroxynitrite formation during ischemia-reperfusion contributes to cerebral injury.
Alonso
David
D
Department of Neuroanatomy and Cell Biology, Instituto de Neurobiología "Santiago Ramón y Cajal," CSIC, E-28002 Madrid, Spain.
Serrano
Julia
J
Rodríguez
Ignacio
I
Ruíz-Cabello
Jesús
J
Fernández
Ana Patricia
AP
Encinas
Juan Manuel
JM
Castro-Blanco
Susana
S
Bentura
María Luisa
ML
Santacana
María
M
Richart
Ana
A
Fernández-Vizarra
Paula
P
Uttenthal
Lars Otto
LO
Rodrigo
José
J
D000818:Animals; D015153:Blotting, Western; D002545:Brain Ischemia / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism; D002540:Cerebral Cortex / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D005947:Glucose / Q000172:deficiency*; D000860:Hypoxia / Q000201:enzymology* / Q000473:pathology / Q000503:physiopathology; D007150:Immunohistochemistry; D008279:Magnetic Resonance Imaging; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology* / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar / Q000033:anatomy & histology / Q000378:metabolism*; D015427:Reperfusion Injury / Q000201:enzymology / Q000473:pathology / Q000503:physiopathology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase; D005947:Glucose; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester