Juan C. Alonso López

Neuronal and inducible nitric oxide synthase expression and protein nitration in rat cerebellum after oxygen and glucose deprivation

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Adrenomedullin expression is up-regulated by ischemia-reperfusion in the cerebral cortex of the adult rat.

Changes in the pattern of adrenomedullin expression in the rat cerebral cortex after ischemia-reperfusion were studied by light and electron microscopic immunohistochemistry using a specific antibody against human adrenomedullin (22-52). Animals were subjected to 30 min of oxygen and glucose deprivation in a perfusion model simulating global cerebral ischemia, and the cerebral cortex was studied after 0, 2, 4, 6, 8, 10 or 12 h of reperfusion. Adrenomedullin immunoreactivity was elevated in certain neuronal structures after 6-12 h of reperfusion as compared with controls. Under these conditions, numerous large pyramidal neurons and some small neurons were intensely stained in all cortical layers. The number of immunoreactive pre- and post-synaptic structures increased with the reperfusion time. Neurons immunoreactive for adrenomedullin presented a normal morphology whereas non-immunoreactive neurons were clearly damaged, suggesting a potential cell-specific protective role for adrenomedullin. The number and intensity of immunoreactive endothelial cells were also progressively elevated as the reperfusion time increased. In addition, the perivascular processes of glial cells and/or pericytes followed a similar pattern, suggesting that adrenomedullin may act as a vasodilator in the cerebrocortical circulation. In summary, adrenomedullin expression is elevated after the ischemic insult and seems to be part of CNS response mechanism to hypoxic injury.

Postnatal changes in the nitric oxide system of the rat cerebral cortex after hypoxia during delivery

The impact of hypoxia in utero during delivery was correlated with the immunocytochemistry, expression and activity of the neuronal (nNOS) and inducible (iNOS) isoforms of the nitric oxide synthase enzyme as well as with the reactivity and expression of nitrotyrosine as a marker of protein nitration during early postnatal development of the cortex. The expression of nNOS in both normal and hypoxic animals increased during the first few postnatal days, reaching a peak at day P5, but a higher expression was consistently found in hypoxic brain. This expression decreased progressively from P7 to P20, but was more prominent in the hypoxic group. Immunoreactivity for iNOS was also higher in the cortex of the hypoxic rats and was more evident between days P0 and P5, decreasing dramatically between P10 and P20 in both groups of rats. Two nitrated proteins of 52 and 38 kDa, were also identified. Nitration of the 52-kDa protein was more intense in the hypoxic animals than in the controls, increasing from P0 to P7 and then decreasing progressively to P20. The 38-kDa nitrated protein was seen only from P10 to P20, and its expression was more intense in control than in the hypoxic group. These results suggest that the NO system may be involved in neuronal maturation and cortical plasticity over postnatal development. Overproduction of NO in the brain of hypoxic animals may constitute an effort to re-establish normal blood flow and may also trigger a cascade of free-radical reactions, leading to modifications in the cortical plasticity.