Research reportPostnatal changes in the nitric oxide system of the rat cerebral cortex after hypoxia during delivery
Introduction
Nitric oxide (NO) is a regulatory biological substance synthesized in numerous types of cells by different isoforms of the enzyme nitric oxide synthase (NOS). Three isoforms of this enzyme catalyse the oxidation of the terminal guanidine nitrogen of l-arginine, yielding equimolar amounts of NO and citrulline [73]. Two of these isoforms, the neuronal (nNOS) and the endothelial (eNOS), have been described as constitutive proteins, while the third is classically termed inducible (iNOS) [38], [60], [63], [69], [74], [76], [77], [80], [81], [102], [105]. The presence of these different NOS isoforms has been demonstrated in numerous species, including mammals [74], [93], [94], [95], fish [35], and invertebrates [33], [34], [41], [71], [72], [113]. Neuronal NOS is a Ca2+-dependent enzyme and its expression in the central and peripheral nervous systems [15], [18], [17], [61], [93], [94], [104] has been previously elucidated. On the other hand, iNOS is Ca2+-independent and its expression is induced by immunological or inflammatory stimuli. This enzyme has been identified in macrophages, mast cells, lymphocytes, neutrophils, platelets, hepatocytes, vascular smooth muscle, tumours, mesangial and endothelial cells [22], [23], [45], [70], [74], [92], [99], [115], astrocytes and neurons of the central nervous system [75].
The main physiological activity of NO is involved in several signal transduction pathways, giving this gaseous molecule a role in the regulation of important activities in the central nervous system throughout life [73]. Transcripts for all three NOS isoforms (neuronal, endothelial, and inducible) are present in the rat brain during embryonic postnatal stages [58], and it is known that nNOS is present during the embryonic period in the cerebral cortex from E11 in rats [101] and from E16 in mice [30]. Rat cortical plates show nNOS activity and mRNA expression from early embryonic development [19], [20], [66], [85], and this activity decreases during postnatal development [16], [42], [82], [90], [117]. The synthesis of NO in cerebral postnatal development has also been studied by biochemical analysis of NOS activity [44], [48], [57], [68].
The NO system might play a prominent role in determining the cortical architecture, participating in the development of the visual system [25], [43], [46], [114], [116], [121], and of olfactory synaptic connections [97].
Between embryonic day 17 (E17) and postnatal day 0 (P0), iNOS is detectable at a low level in comparison with the high expression of nNOS [96], [100]. The iNOS increases from P1 to P6, and then declines from this stage to very low expression during the adult period, while nNOS, as a compensatory response [62], maintains comparatively high immunocytochemical expression throughout adult life [93].
Hypoxic exposure during the perinatal period is a common cause of brain damage, and NO appears to be critical in the pathophysiology of this process [27], [51] as well as in ischaemic brain injury. Also, NO can function as either a neuroprotective agent through its vasodilator effect or as a neurotoxic agent. These effects have been studied in various animal models and species, including the rat [21], [48], [79]. The neurotoxic effect of NO results from an excessive burst of NO following intense neural stress.
It has also been demonstrated in the adult rat brain that the NO produced by NOS is directly involved in cerebrovascular lesions through rapid upregulation of nNOS, its mRNA [120], and, subsequently, NO production. In the cortical areas affected by cerebrovascular insults, the effect of NO may be related to the presence of immunoreactive neurons close to cerebral arteries [37], [54]. It has been reported that the administration of a competitive N-methyl-d-aspartate antagonist significantly reduces infarcted brain volume [87]. Moreover, in mutant mice deficient in nNOS activity, medial carotid artery occlusion also decreases the volume of infarcted tissue [53].
Excessive synthesis of NO injures the adjacent tissues by its rapid reaction with superoxide radicals (O2−) to yield peroxynitrite (ONOO−) [26], [27], [79], which undergoes protonation, isomerization, and decomposition reactions [10], [11], [89]. The amino acid tyrosine is the main protein target for nitration, and nitrotyrosine residues have been demonstrated after experimental carbon monoxide poisoning in the rat brain [56] as well as in the cortex of aging rats [109], suggesting the formation of peroxynitrite and its action on cell proteins in the brain. Lesions in the rat somatosensory cortex have also shown nitrotyrosine immunoreactivity in blood vessels close to the lesion as well as in the ipsilateral hippocampus and thalamus [12].
In this comparative work, immunocytochemical and biochemical procedures were used to determine the immunoreactive distribution, expression and activity of nNOS and iNOS, and the reactivity to nitrotyrosine during the early postnatal development of cerebral cortex, using brains of normal rats and rats submitted to in utero hypoxia during delivery.
Section snippets
Experimental model of hypoxia in newborn rats
Pregnant albino Wistar rats (250–300 g) at 21 days of gestation, just before delivery, were decapitated and the bodies kept at 37 °C. The foetuses were left inside the uterus for 20 min in order to generate hypoxic conditions. The pups were removed by hysterectomy, resuscitated by external thoracic stimulation, and then placed under the protection of a wet-nurse mother rat during postnatal development. For comparison purposes, the control group consisted of pregnant rats that were allowed a
Postnatal day 0–20 (P0–P20) control rats
Immunoreactivity to nNOS in control newborn rats, P0 postnatal day, was found in neurons with bipolar/fusiform morphology, which pervaded the frontal, parietal and agranular-insular cortices. The cell bodies distributed mainly in the deep region of cortical plate contained a nNOS-positive thin cytoplasmic ring, unstained nucleus, and usually a long and varicose apical process and occasionally some short basal processes. The apical processes, without collaterals crossed the entire cortical plate
Discussion
This study was performed during the first phase of the postnatal period (P0–P20), with two groups of rats born normally and after 20 min of hypoxia in utero during delivery. The study comparatively analyses the involvement of nNOS, iNOS, and nitrotyrosine as markers of protein nitration in the postnatal cortical cytoarchitectonic development.
The immunohistochemical findings for nNOS, iNOS, and nitrotyrosine, were quantified by Western blotting and NO production using biochemical analysis of NOS
Acknowledgements
This work was supported by Grants DGYCIT PM 98-0126-C02-01, Comunidad de Madrid 08.5/0054/2000-1 and Caixa Foundation 99/077-00. We thank E. Sánchez and A. Sandonis for expert technical assistance and D. Nesbitt for his help with manuscript preparation.
References (121)
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium
Neuron
(1994) - et al.
Nitric oxide synthase protein and mRNA are discretely localized in neuronal population of the mammalian CNS together with NADPH-diaphorase
Neuron
(1991) - et al.
Nitric oxide synthase activity in the hippocampus, frontal cerebral cortex, and cerebellum of the guinea pig: ontogeny and in vitro ethanol exposure
Alcohol
(1995) - et al.
Nitric oxide neurotoxicity
J. Chem. Neuroanat.
(1996) - et al.
Ontogenesis of NADPH-diaphorase neurons in the mouse forebrain
Neurosci. Lett.
(1993) - et al.
Gap junctions in the brain: where, what type, how many and why?
Trends Neurosci.
(1993) - et al.
Nitric oxide synthesis and action in an invertebrate brain
Brain Res.
(1993) - et al.
Expression of mouse brain soluble guanylyl cyclase and NO synthase during ontogeny
Dev. Brain Res.
(1994) - et al.
Postnatal development of NADPH-diaphorase activity in the superior colliculus and the ventral lateral geniculate nucleus of the rat
Dev. Brain Res.
(1993)
Cytosolic and membrane-bound cerebral nitric oxide synthase activity during hypoxia in cortical tissue of newborn piglets
Neurosci. Lett.
Increase in nitric oxide in the hypoxic–ischemic neonatal rat brain and suppression by 7-nitroindazole and aminoguanidine
Eur. J. Pharmacol.
Bright and dark sides of nitric oxide in ischemic brain injury
Trends Neurosci.
Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase
Arch. Biochem. Biophys.
Effect of hypoxia and reoxygenation on regional activity of nitric oxide synthase in brain of newborn piglets
Neurosci. Lett.
Patterns of nitric oxide synthase at the messenger RNA and protein levels during early rat brain development
Neuroscience
Nitric oxide: cytotoxicity versus cytoprotection –how, why, when and where?
Nitric Oxide
Presence of nitric oxide synthase activity in the neurons of the rat embryonal cerebrum
Neurosci. Lett.
Nitric oxide synthase structure and mechanism
J. Biol. Chem.
NADPH-diaphorase/nitric oxide synthase in the central nervous system of spiders (Arachnida: Araneida)
Neurosci. Lett.
Enzymatic characterization of recombinant murine inducible nitric oxide synthase
Eur. J. Pharmacol.
Regulation of biosynthesis of nitric oxide
J. Biol. Chem.
Immunohistochemical nitrotyrosine distribution in neonatal rat cerebrocortical slices during and after hypoxia
Brain Res.
Neuronal nitric oxide synthase expression in neuronal cell differentiation
Neurosci. Lett.
Nitric oxide induces cultured cortical neuron apoptosis
Neurosci. Lett.
Efficacy of D-CPPene, a competitive N-methyl-d-aspartate antagonist in focal cerebral ischemia in the rat
Neurosci. Lett.
Distribution of nitric oxide synthase in the esophagus of the cat and monkey
J. Auton. Nerv. Syst.
The cessation of mitosis in the central nervous system of the albino rat
J. Comp. Neurol.
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
J. Comp. Neurol.
Preconditioning regulation of bcl-2 and p66shc by human NOS I enhances tolerance to oxidative stress
FASEB J.
Cajal cells of the rabbit cerebral cortex
Experientia
Cyclic CMP-dependent inhibition of acid sphigomyelinase by nitric oxide: an early step in production against apoptosis
Cell Death Differ.
Peroxynitrite: mediator of the toxic action of nitric oxide
Acta Biochem. Pol.
Ischaemic injury mediator
Nature
Oxidative damage and tyrosine nitration from peroxynitrite
Chem. Res. Toxicol.
Nitric oxide, superoxide and peroxynitrite: the good, the bad and the ugly
Am. J. Physiol.
Extensive nitration of protein tyrosines in human atherosclerosis detected by immunocytochemistry
Biol. Chem.
Kinetics of superoxide dismutase and iron-catalyzed nitration of phenolics by peroxynitrite
Arch. Biochem. Biophys.
Transient changes in the presence of nitric oxide synthases and nitrotyrosine immunoreactivity after focal cortical lesions
Neuroscience
Neurons in layer I of the developing occipital cortex of the rat
J. Comp. Neurol.
Isolation of nitric oxide synthase: a calmodulin requiring enzyme
Proc. Natl. Acad. Sci. USA
Localization of nitric oxide synthase indicating a neuronal role for nitric oxide
Nature (London)
Regulation of neuronal nitric oxide synthase through alternative transcripts
Dev. Neurosci.
Mechanisms involved in the neuroprotective activity of a nitric oxide synthase inhibitor during focal cerebral ischemia
J. Neurochem.
Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells
FEBS Lett.
Further evidence of the presence of constitutive and inducible nitric oxide synthase isoforms in human platelets
J. Cardiovasc. Pharmacol.
The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications
Free Radic. Res.
Transient expression of NADPH-diaphorase in the lateral geniculate nucleus of the ferret during early postnatal development
J. Comp. Neurol.
Nitric oxide and focal cerebral ischemia multiplicity of actions and diverse outcome
Cerebrovasc. Brain Metab. Rev.
Nitric oxide actions and pathological roles
Neuroscientist
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