The nitric oxide synthase expression of rat cortical and hippocampal neurons changes after early lead exposure.

The effect of lead exposure in nitric oxide synthase containing neurons (nNOS) within rat cortex and hippocampus was studied. Lead administration (1 g% lead acetate in drinking water) was commenced prior to mating and continued until 30 postnatal (PN) days. Immunohistochemical studies using antibody to nNOS showed, after lead treatment at PN21-PN30, a reduction in neuronal size and optical density (OD) of nNOS+ cells. In both regions, non-pyramidal immunoreactive neurons exhibited smaller soma size and less developed dendrites. A significant difference in cell areas and OD of lead exposed versus control rats and no variation in the number of nNOS+ neurons was seen. Morphological modifications after early lead exposure, induced nNOS reduction in NOS expressing neurons thereby interfering in NO synthesis.

Short-term changes in NADPH-diaphorase reactivity in rat brain following perinatal asphyxia. Neuroprotective effects of cold treatment.

Perinatal asphyxia (PA) produces changes in nitric oxide synthase (NOS) activity in neuronal and endothelial cells of the striatum and neocortex. The changes were examined using a histochemical NADPH-diaphorase (NADPH-d) staining method. Newborn rats were exposed to severe PA at 37 degrees C and other groups were subjected to severe PA under hypothermic condition (15 degrees C) for 20 or 100 min, respectively. Quantitative image analysis was performed on the striatum and neocortex in order to count cell number of reactive neurons and to compare the pattern of staining between the different groups of animals. Severe asphyctic pups showed an important neuronal loss in striatum and neocortex that was reduced by hypothermia. NADPH-d(+) neurons with reactive processes were found in the lateral zone of the striatum and neocortex in asphyctic pups. Controls and hypothermic striatum showed rounded cells without reactive process, while no cells were stained in cortex. There was also an increase in NADPH-d activity in endothelial cells in severe asphyctic pups in striatum and neocortex vs control and hypothermically treated animals. Our data evidenced that an inappropriate activation of NOS in neuronal and endothelial cells induced by PA is related to neuronal injury. Hypothermia inhibits neuronal injury and may be a valuable neuroprotective agent.

Prenatal and postnatal lead exposure induces 70 kDa heat shock protein in young rat brain prior to changes in astrocyte cytoskeleton.

In previous studies we found that chronic postnatal (PN) lead exposure [1 g% (w/v)] induced astroglial hypertrophy in rat hippocampus. Since astrocytic responses change upon the stage during which exposure occurs, astroglial reactions in cerebral cortex and hippocampus of young animals were studied and compared when exposure began during development. Lead-treatment started 90 days prior to mating, and was maintained during gestation and after birth up to PN160. Alterations observed from PN21 to PN140 were assessed by antibodies to the 70kDa heat shock proteins (hsp), glial fibrillary acidic protein (GFAP) and vimentin (VIM) using immunohistochemistry, transmission electron microscopy (TEM), and computer assisted image analysis. The induction of hsp was seen from PN21 to PN45 in non-pyramidal neurons and astrocytes, and at the same time, astroglial swelling was noticed. After PN45 the resolution of this edema coincided with an increase of gliofilaments and GFAP and VIM immunoreaction (PN60-PN90). Recovery of VIM expression persisted after PN120 in the hilus; meanwhile, lipofuscin-like bodies appeared in neurons and astrocytes. Lead exposure during rapid brain growth induced hsp after weaning in neurons and astrocytes prior to astrocyte cytoskeletal changes. Astroglial and neuronal alterations could modify complex neuron-glia interactions, disturbing brain function in consequence.

Long term changes in NADPH-diaphorase reactivity in striatal and cortical neurons following experimental perinatal asphyxia: neuroprotective effects of hypothermia.

Nitric oxide (NO) is known to be involved in the neuropathological mechanisms triggered by excitatory aminoacids. NO(+) neurons in the brain may be detected histochemically by nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemical technique, as the latter readily labels NO synthase in the central nervous system (CNS). NADPH-d stained striatal and cortical sections were studied in 6-month-old male Sprague-Dawley rats exposed to perinatal asphyxia (PA) at 37 degrees C, as well as in animals subjected to PA plus hypothermia treatment at 15 degrees C. Quantitative image analysis was performed to compare the staining pattern in the various groups. NADPH-d(+) neurons in striatum and cortex from subsevere and severe asphyctic animals showed a significant increase in soma size and in dendritic processes versus controls and hypothermia-treated rats. These findings indicate that chronic NO changes are involved in postischemic striatal and cortical alterations induced by PA that may be prevented by hypothermia.

Ultrastructural changes in nitric oxide synthase immunoreactivity in the brain of rats subjected to perinatal asphyxia: neuroprotective effects of cold treatment.

Striatal and cortical neurons containing nitric oxide synthase (NOS) were studied in adult rats subjected to different periods of perinatal asphyxia (PA) using immunohistochemistry at both light microscopy (LM) and electron microscopy (EM). Another group was subjected to PA + hypothermia to study its neuroprotective effect. Quantitative image analysis was performed on the striatum and neocortex in order to count the number of immunoreactive neurons and to compare the pattern of staining between the different groups. Six-month-old rats that suffered subsevere and severe PA demonstrated, at LM, cytomegaly of the striatal and neocortical neurons containing NOS. Control and hypothermic neurons were more weakly immunostained than PA neurons. Subsevere and severe asphyctic rats showed an important neuronal loss that was reduced by hypothermic treatment. The PA group disclosed, at EM, dense electronic bodies distributed in terminals surrounding synaptic vesicles and in dendrites. Non-NOS-containing neurons showed signs of degeneration, such as dark cytoplasm and shrunken nuclei. Surrounding the blood vessels, we observed a clear edema. The immunolabeling in hypothermic rats resembled that observed in controls. These data suggest that subsevere and severe PA induces chronic changes in the neuronal content of NOS in the striatum and neocortex. Degeneration observed in neurons surrounding cytomegalic NOS-containing cells may be due to the excess of NO in their environment. Moreover, the chronic alterations produced by PA seem to be prevented by hypothermia.

Striatal cytomegalic neurons containing nitric oxide are associated with experimental perinatal asphyxia: implication of cold treatment.

Neuropathological mechanisms triggered by excitatory aminoacids are known to involve nitric oxide (NO). Neurons containing NO are histochemically reactive to nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), which labels NO synthase in CNS. Sprague-Dawley male rats subjected to perinatal asphyxia (PA) at 37 degrees C, and PA plus 15 degrees C hypothermia were evaluated when 6 months old by NADPH-d histochemical reaction. Computarized image analysis was used for quantification of stained sections. NADPH-d neurons in striatum from subsevere and severe PA showed a significant increment in soma size and dendritic process length versus control and hypothermic treated rats. Post-ischemic damage neurons are therefore involved in NO changes induced by PA that may be prevented by hypothermia treatment.

Prolonged lead exposure modifies astrocyte cytoskeletal proteins in the rat brain.

The time-course and the regional astrocyte responses were studied during 15 months of continuous lead-exposure. Rat pups were exposed from postnatal day 1 (P1 group) and day 7 (P7 group) through the maternal milk, (1g% lead acetate solution in the drinking water). Following weaning lead-exposed offspring were treated during 15 months. Immunohistochemical staining for glial fibrillar acidic protein (GFAP) and vimentin were used to evaluate astrocyte response. In coincidence with previous results, after 2-3 months of treatment, we observed in both groups a transient increase of GFAP immunoreactivity in hippocampal and cerebellar astrocytes (first stage). After 4-12 months (second stage), the hypertrophy declined to near control levels despite the persistence of high blood lead-levels; meanwhile, lipofuscin-like bodies appeared in neuronal and glial cells. After one year of treatment, the immunostaining of homologous sections showed an increase of GFAP reactivity and the presence of vimentin + cells in the upper and the lower limb, and in the hilus of the fascia dentata. GFAP and vimentin astrocytic response extended to the adjacent cerebral cortex after 14 months. Although both cells showed a similar aspect, vimentin + cells showed a smaller area and a restricted arrangement. However, in the cerebellum the hypertrophy of vimentin positive astrocyte and Bergmann fibers was confined to the white matter as observed in the first stage. Astrocyte alterations, the recovery of vimentin expression and the appearance of lipofuscin-like bodies induced by prolonged lead-exposure suggest modifications in neuronal microenvironment, and might accelerate age-dependent changes in CNS.

Chronic lead exposure induces astrogliosis in hippocampus and cerebellum.

The aim of this study was to characterize the cytoskeletal intermediate filaments, glial fibrillary acidic protein (GFAP), and vimentin in normal and lead treated rats, and to compare the astroglial response in the cerebellum and the hippocampus -two regions with great susceptibility to the toxic effects of lead. Experiments combined light and electron microscopy immunohistochemistry using antibodies to GFAP and to vimentin, and conventional transmission electron microscopy techniques. Chronic lead administration was provided through the drinking water (1 g% lead acetate solution) and started when pups were 7 days old through the mother's milk. Following weaning lead intoxicated offspring were continuously exposed during 9 months, and sacrificed, with their corresponding controls, by perfusion-fixation at 30, 60, 75, 90, 180 and 270 days of lead exposure. After 60 and 90 days of treatment, hypertrophic astrocytes were observed in the cerebellum and hippocampus. Additionally, in the same time-period more GFAP immunolabelled astrocytes were detected in the cerebellum but not in the hippocampus. These qualitative observations were confirmed by computerized image analysis quantification. This effect was transient, even though the lead treatment was prolonged for 9 months and the blood-lead levels remained high after 30 days of the lead-exposure. After 90 days of lead administration, hypertrophic astrocytes started to decline and a gradual increment in the number of dense bodies, lipofuscin-like, was evidenced in astrocytes, neurons, pericytes and microglial cells. The data suggest that chronic lead exposure induces an astrocytic reaction as a result of a direct action of lead on astroglial cells or as a response to underlying neural damage.

Chronic lead exposure induces alterations on local circuit neurons.

The effect of chronic lead exposure in neonatal rats was examined, particularly on hippocampal local circuit neurons (LCN). Chronic lead was administered to rat pups in the first week of life through the mother milk. During the lactation period the mother only drank water containing 1 g% lead acetate (w/v) -subclinical dose. Following weaning, the young rats were treated for 3 months with the same concentration of lead-adulterated water. Electron microscopy studies were made at 30, 45, 60, 75 and 90 days of age. After 2 months of lead exposure, irregular outline and dark-condensed cytoplasm in non-pyramidal neurons, in basket and in granule cells were found. Within the neuropil, some beaded and smooth dendrites, as well as some axons and synaptic terminals, appeared condensed and darker than the surrounding elements. Coincident with the LCN alterations, astrocytes showed an increment of glial filaments. These results suggested that the functional activity of LCN could be modified by the observed changes. Such ultrastructural modifications could be a morphological substrate for the subtle neuropsychological deficits of rats chronically exposed to relatively low concentration of lead.

Astroglial alterations in rat hippocampus during chronic lead exposure.

The present study was performed in order to follow the response of astroglial cells in the rat hippocampus to chronic low-level lead exposure. The experiments combined immunohistochemistry using anti-glial fibrillary acidic protein (GFAP) antibody and conventional transmission electron microscopy (EM). Chronic administration with drinking water [1 g% w/v (subclinical dose) of lead acetate dissolved in distilled water] was started through the mother's milk when pups were 7 days old. Following weaning, experimental offspring were treated for 3 months with the same concentration of adulterated water. The group of intoxicated animals and their controls were sacrificed by perfusion-fixation at 30, 60, and 90 days of exposure. After 60 days of lead treatment, staining of GFAP-positive cells demonstrated an astroglial transformation from the quiescent to the reactive state, characterized by an increase in GFAP. In control rats no changes in GFAP immunostaining were observed. The intensity of the astroglial response was enhanced after 90 days of lead intoxication, showing an increment of GFAP immunoreactivity. Quantification of these changes was made by computerized image analysis, confirming that the sectional areas of the astroglia in lead-exposed animals were larger than those in controls. These results are consistent with the ultrastructural alterations. Simultaneously with the increment in gliofilaments, intranuclear inclusions were seen in some astrocytes. The mechanisms by which lead affects astrocytes are unknown. Probably the astroglial changes induced by lead intoxication produce microenvironmental modifications that may disturb the neuronal function.

Chronic lead exposure induces alterations on local circuit neurons.

The effect of chronic lead exposure in neonatal rats was examined, particularly on hippocampal local circuit neurons (LCN). Chronic lead was administered to rat pups in the first week of life through the mother milk. During the lactation period the mother only drank water containing 1 g

lead acetate (w/v) -subclinical dose. Following weaning, the young rats were treated for 3 months with the same concentration of lead-adulterated water. Electron microscopy studies were made at 30, 45, 60, 75 and 90 days of age. After 2 months of lead exposure, irregular outline and dark-condensed cytoplasm in non-pyramidal neurons, in basket and in granule cells were found. Within the neuropil, some beaded and smooth dendrites, as well as some axons and synaptic terminals, appeared condensed and darker than the surrounding elements. Coincident with the LCN alterations, astrocytes showed an increment of glial filaments. These results suggested that the functional activity of LCN could be modified by the observed changes. Such ultrastructural modifications could be a morphological substrate for the subtle neuropsychological deficits of rats chronically exposed to relatively low concentration of lead.

Axon-glia relationships in crab nerves.

The nerves of the walking legs in the crab Lebidoclea grammania were studied by electron microscopy. Particular atthetion was directed to the structure of the connective tissue, its arrangement within the axon sheaths and its association with the glial cells. The connective tissue of the neural lamella of the giant axons and the fascicles is formed by collagen fibrils aand bands of mucopolysaccharides. Prolongations of the neural lamella divide the fascicles into bundles of contiguous axons, groups of loosely sheathed axons and nerve fibres wrapped by layers of glial cell processes alternating with layers of connective tissue. The glial cell processes close to the axons contained numerous microtubules whereas glycogen granules predominated in the more peripheral processes. These observations suggest that the connective tissue and glial cell processes forming the envelopes of the axons together participate in the maintenance of the microenvironment around axons.