María Luisa Del Moral

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.

LIGHT MICROSCOPIC QUANTIFICATION OF MORPHOLOGICAL CHANGES DURING AGING IN NEURONS AND GLIA OF THE RAT PARIETAL CORTEX

Different changes in neuronal and glial population of the aging brain have been described; however, the degree and extent of these changes are controversial. This study evaluates the quantitative and cytomorphometric effects of aging on neuronal and glial populations in the parietal cortex of the rat.

QUANTITATIVE AND ULTRASTRUCTURAL CHANGES IN GLIA AND PERICYTES IN THE PARIETAL CORTEX OF THE AGING RAT

The frequency of astrocytes, microglia plus oligodendrocytes, and pericytes displaying nuclei was analyzed and quantified in 160‐μm‐wide strips of the parietal cortex (Par1 region) from young and aged Wistar rats. The study was performed on two groups of rats aged 3–4 and 32–36 months. Quantifications of the glial cell types and pericytes were made in 1‐μm‐thick sections stained with toluidine blue. Ultrathin sections were also made to analyze the ultrastructural features of these cells during aging. Astrocytes and pericytes increased in number by about 20% and 22%, respectively, with age. These increases were most significant in layers II–IV and V for both cellular types. Clusters of astrocytes were common in these layers of aging rats. The ultrastructural analysis also indicated changes in all cell types that stored inclusions and vacuoles with age, which were particularly abundant in microglial cells. End‐feet astrocytes and pericytes surrounding the vascular wall also contained vacuoles and inclusions, and consequently the vascular wall increased in thickness. In conclusion, the aging process increased astrocyte and pericyte populations, but not microglia plus oligodendrocyte populations, in the rat parietal cortex. Although no significant change in nuclear size could be observed in any cell type, all glial cells as well as pericytes underwent morphological ultrastructural changes. These modifications may result from the need to correct possible homeostatic imbalances during aging. Microsc. Res. Tech. 43:34–42, 1998.

Immunohistochemical localisation of neuronal nitric oxide synthase in the rainbow trout kidney.

The distribution of nitrergic nervous structures in the trout kidney was studied by peroxidase-linked ABC immunostaining procedures using a polyclonal antibody raised against the neuronal isoform of nitric oxide synthase. The nitrergic plexus reaches the kidney along the vasculature, mainly running with the postcardinal vein where nitrergic fibres, microganglia like cellular clusters and isolated neurones were detected. The atubular head-kidney only showed isolated nitrergic fibres close to the larger arteries. On the other hand, the collecting tubules, collecting ducts, large arteries and glomerular arterioles of the tubular middle and posterior trunks were innervated by nitrergic fibres even though immunoreactive neurones were also observed in close apposition to some tubular elements and large arteries. These results suggest that, according to morphofunctional differences between the fish and mammalian kidneys, nitrergic neural structures may be involved in the control of particular renal functions in the rainbow trout.

Study of the nitric oxide system in the rat cerebellum during aging

BackgroundThe cerebellum is the neural structure with the highest levels of nitric oxide, a neurotransmitter that has been proposed to play a key role in the brain aging, although knowledge concerning its contribution to cerebellar senescence is still unclear, due mainly to absence of integrative studies that jointly evaluate the main factors involved in its cell production and function. Consequently, in the present study, we investigate the expression, location, and activity of nitric oxide synthase isoenzymes; the protein nitration; and the production of nitric oxide in the cerebellum of adult and old rats.ResultsOur results show no variation in the expression of nitric oxide synthase isoforms with aging, although, we have detected some changes in the cellular distribution pattern of the inducible isoform particularly in the cerebellar nuclei. There is also an increase in nitric oxide synthase activity, as well as greater protein-nitration levels, and maintenance of nitrogen oxides (NOx) levels in the senescent cerebellum.ConclusionsThe nitric oxide/nitric oxide syntahses system suffers from a number of changes, mainly in the inducible nitric oxide synthase distribution and in overall nitric oxide synthases activity in the senescent cerebellum, which result in an increase of the protein nitration. These changes might be related to the oxidative damage detected with aging in the cerebellum.

Nitrergic innervation of the cat liver

The aim of this work was to study the nitrergic innervation in the liver of the cat using immunocytochemical procedures. At the hepatic hilus, a rich plexus of neuronal nitric oxide synthase immunoreactive (nNOS-IR) nerve fibers and ganglia was detected around the interlobular branch of the bile duct. nNOS-IR nerve fibers were observed running with the components of the intralobular portal triads located close to the hepatic hilus, as well as with a few vessels and ducts of the deeper parenchyma. These latter fibers, beside others located in Glissons capsule, occasionally showed short ramifications entering the parenchyma itself. The present results suggest that, in the cat liver, nNOS is involved in the autonomic control of hepatic blood flow, with a limited role in the regulation of hepatobiliary excretory activity and hepatocellular metabolic function.

Does inducible NOS have a protective role against hypoxia/reoxygenation injury in rat heart?

PURPOSE The present study analyzes the role of the nitric oxide (NO) derived from inducible NO synthase (iNOS) under cardiac hypoxia/reoxygenation situations. METHODS For this, we have designed a follow-up study of different parameters of cell and tissue damage in the heart of Wistar rats submitted for 30 min to acute hypobaric hypoxia, with or without prior treatment with the selective iNOS inhibitor N-(3-(aminomethyl)benzyl) acetamidine or 1400W (10 mg/kg). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analyzing NO production (NOx), lipid peroxidation, apoptosis, and protein nitration expression and location. This is the first time-course study which analyzes the effects of the iNOS inhibition by 1400W during hypoxia/reoxygenation in the adult rat heart. RESULTS The results show that when 1400W was administered before the hypoxic episode, NOx levels fell, while both the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. Levels of nitrated proteins expression fell only at 12 h post-hypoxia. CONCLUSIONS The inhibition of iNOS raises the peroxidative and apoptotic level in the hypoxic heart indicating that this isoform may have a protective effect on this organ against hypoxia/reoxygenation injuries, and challenging the conventional wisdom that iNOS is deleterious under these conditions. These findings could help in the design of new treatments based on NO pharmacology against hypoxia/reoxygenation dysfunctions.

Inducible NOS inhibitor 1400W reduces hypoxia/re-oxygenation injury in rat lung

Abstract Nitric oxide (NO•) from inducible NO• synthase (iNOS) has been reported to either protect against, or contribute to, hypoxia/re-oxygenation lung injury. The present work aimed to clarify this double role in the hypoxic lung. With this objective, a follow-up study was made in Wistar rats submitted to hypoxia/re-oxygenation (hypoxia for 30 min; re-oxygenation of 0 h, 48 h, and 5 days), with or without prior treatment with the selective iNOS inhibitor 1400W (10 mg/kg). NO• levels (NOx), lipid peroxidation, apoptosis, and protein nitration were analysed. This is the first time-course study which investigates the effects of 1400W during hypoxia/re-oxygenation in the rat lung. The results showed that the administration of 1400W lowered NOx levels in all the experimental groups. In addition, lipid peroxidation, the percentage of apoptotic cells, and nitrated protein expression fell in the late post-hypoxia period (48 h and 5 days). Our results reveal that the inhibition of iNOS in the hypoxic lung reduced the damage observed before the treatment with 1400W, suggesting that iNOS-derived NO• may exert a negative effect on this organ during hypoxia/re-oxygenation. These findings are notable, since they indicate that any therapeutic strategy aimed at controlling excess generation of NO• from iNOS may be useful in alleviating NO•-mediated adverse effects in hypoxic lungs.

The nitrergic autonomic innervation of the liver.

Using immunohistochemical approaches, the nitrergic innervation of the liver has been studied in mammals and in fish. The morphofunctional relationships described here indicate a high degree of evolutionary conservation of the hepatic nitrergic transmission and its possible involvement in the neural control of the hepatic function in health and disease.

Age modulates the nitric oxide system response in the ischemic cerebellum.

To determine whether age influences the nitric oxide system response to ischemia in the cerebellum, we have analyzed the levels of nitrogen oxides (NOx) and the expression of the different nitric oxide synthase isoforms (NOS) in mature adult (4-5 months old) and aged rats (24-27 months old) subjected to a transient global ischemia/reperfusion (I/R) model. We also analyzed the nitrated proteins and the glial fibrillary acidic protein (GFAP) expression. NOx concentration in adult rats, which more than doubled the values found in the aged rats, decreased after the ischemia and reperfusion. However, in the aged animals, these NOx levels did not significantly change after I/R. Constitutive isoforms were first down-regulated in the ischemic period, in both adult and aged animals. However, after 6 h of reperfusion, these isoforms were up-regulated, but only in aged rats. After I/R, iNOS was up-regulated in adults but down-regulated in the aged rats. Hence, after an episode of transient global ischemia and reperfusion, the aged cerebellum maintains a balanced NO production, silencing the iNOS isoform and inducing a weak expression of nNOS and eNOS; this allows NO physiological functions while avoiding possible undesirable effects such as the nitrative damage or astrocyte activation.