Changes in reactive oxygen species (ROS) production in rat brain during global perinatal asphyxia: an ESR study.

A large body of evidence suggests that the production of reactive oxygen species (ROS) can play an important role in ischemic neuronal injury. However any studies has been performed in hypoxic conditions. In the present experiments we studied using electron spin resonance (ESR) techniques the ROS release in neostriatum of newborn rats subjected to acute perinatal asphyxia (PA) followed by various periods of reoxygenation. Pregnant rats' uteri still containing foetuses were taken out and subjected to PA by immersion in a 37 degrees C water bath during the following periods of time: 5, 10, 15, 19 and 20 min. After performing PA, animals were recovered and ROS measured after 0, 5, 15, 30 or 60 min of reoxygenation. Then, pups were sacrificed, their neostriatum removed and homogenised with N-tert.-butyl-alpha-phenylnitrone (PBN) and diethylenetriamine-pentacetic acid (DPTA) in phosphate-buffered saline (PBS) and the formed complexes were extracted with ethyl acetate an analysed using an X-band ESR spectrometer. A significant release of ROS was detected at 19 and 20 min of PA after 5 min of reoxygenation. These data provide strong evidence that ROS could be involved in neuronal damage during PA.

Neuroglial interactions in a model of para-chlorophenylalanine-induced serotonin depletion.

Serotonin (5HT) is involved in the development and plasticity of the CNS through the release of S-100beta, a glial trophic factor which stabilizes synapses and neuronal cytoskeleton and promotes neuronal development. S-100beta is released from glial cells after activation of glial 5HT(1A) receptors. We present in this paper the effects upon neurons and glia of a 5HT depletion induced by 14 days of treatment with para-chlorophenylalanine (PCPA) in adult rats. S-100beta, 5HT, 5HT-transporter (5HT-T) and neurofilaments (Nf-200 and Nf-68) expressions were studied by immunohistochemistry and image analysis in striatum, hippocampus, parietal and frontal cortex. Immediately after ending PCPA treatment we found increased intracellular S-100beta immunoreactivity in glial cells, reduced 5HT immunolabelling, reduced density of 5HT-T, Nf-200 and Nf-68 fibers and morphological alterations in neuronal cytoskeleton. One week after PCPA treatment S-100beta immunoreactivity decreased towards control levels, 5HT was normalized in dorsal raphe nucleus, but not in innervation areas; 5HT-T, Nf-200 and Nf-68 fiber densities increased but some neuronal cytoskeletal alterations were still present in striatum. Two weeks after PCPA treatment S-100beta had returned to control levels in most studied regions; 5HT immunoreactivity was normalized, meanwhile 5HT-T, Nf-200 and Nf-68 fiber densities increased reaching values over the control level. We propose that S-100beta could be accumulated in glial cells during the 5HT depletion period, to be released once 5HT levels have recovered. Neuronal cytoskeletal alterations and reduced fiber density may be the expression of decreased extracellular availability of S-100beta. Conversely, increased 5HT-T, Nf-200 and Nf-68 expressions, once S-100beta is normalized, may be the biological response to the growth factor release.

Atrial natriuretic peptide effect on NADPH-diaphorase in rat intestinal tract.

Histochemical reaction of NADPH-diaphorase (NOS-NADPH-d) was used to identify NO synthesis. A 30-min 0.1 microg microg/kg/min ANP infusion led to about a 10% and 35% increase in small and large intestine enterocytes stain respectively. This increase was abolished by a bolus of 1 mg/kg L-NAME before ANP infusion in small intestine, and partially abolished it in colon. Incubation of small and large intestine with 0.5 microM ANP increased stain at about 20%. In both tissues the preincubation with 0.1 mM L-NAME abolished the ANP effect. Incubation with 0.1 mM 8-Br-cGMP enhanced staining about 70% and 30% in small and large intestine respectively. Our results show that ANP enhances NOS-NADPH-d activity, suggesting that ANP stimulates NO synthase in enterocytes by L-arginine-NO pathway. 8-Br-cGMP mimicked the effect of ANP described above. Therefore, the guanylate cyclase-coupled natriuretic receptors, NPR-A and NPR-B, probably mediate this ANP effect.

GABA release mechanism in the golden hamster retina.

High K+ medium and glutamate elicited a significant [3H]-GABA release in the golden hamster retina. High K+ -induced GABA release was largely calcium-dependent, while the effect of glutamate was Ca2+ -independent. After replacing Na+ by Li+, glutamate-evoked [3H]-GABA release was abolished, while high K+ -evoked release remained unchanged. The effect of glutamate was completely blocked by DNQX but not by APV. Furthermore, kainate induced [3H]-GABA release, whereas NMDA was ineffective. Assessment of endogenous GABA efflux further confirmed results obtained for [3H]-GABA. GABA-like immunoreactivity was observed in amacrine cells, in neurons localized in ganglion cell layer, as well as in fibers and terminals at the inner plexiform layer. In addition a few horizontal cells showed GABA-like immunolabeling. The present results suggest the existence of at least two pools of GABA in the hamster retina, compatible with both vesicular and carrier-mediated mechanisms of transmitter release, being the amacrine cells the main gabaergic source in this tissue.

Serotonergic ependymal fibres in rat and monkey: light and electron microscopic immunocytochemical study

The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc

Distribution of GABA in the golden hamster retina: An immunocytochemical study

Distribution of the inhibitory neurotransmitter GABA was studied in the golden hamster retina using immunocytochemistry at cellular and subcellular levels. GABA-immunoreactivity was observed in somata of amacrine, displaced amacrine and horizontal cells. GABA immunoreactive fibers were abundant in the inner plexiform layer. Ultrastructural analysis exhibited dense GABA-immunoreactive deposits in amacrine cell somata, processes and terminals. Immunolabelling was also observed in the cytoplasm of horizontal or interplexiform cells and displaced amacrine cells. In every case DAB deposits were observed in the cytosolic compartment, attached to the inner surface of cell membranes and to outer mitochondrial membranes. Immunolabeled terminals predominated in the inner plexiform layer and immunoprecipitates were also observed attached to the outer face of vesicle membranes as well as completely filling synaptic vesicles. Both clear and dense core vesicles were observed. The present results are similar to those obtained in other mammalian species showing GABA immunoreactivity in amacrine, displaced amacrine and horizontal cells

Serotonergic ependymal fibres in rat and monkey: light and electron microscopic immunocytochemical study

The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc

Distribution of GABA in the golden hamster retina: An immunocytochemical study

Distribution of the inhibitory neurotransmitter GABA was studied in the golden hamster retina using immunocytochemistry at cellular and subcellular levels. GABA-immunoreactivity was observed in somata of amacrine, displaced amacrine and horizontal cells. GABA immunoreactive fibers were abundant in the inner plexiform layer. Ultrastructural analysis exhibited dense GABA-immunoreactive deposits in amacrine cell somata, processes and terminals. Immunolabelling was also observed in the cytoplasm of horizontal or interplexiform cells and displaced amacrine cells. In every case DAB deposits were observed in the cytosolic compartment, attached to the inner surface of cell membranes and to outer mitochondrial membranes. Immunolabeled terminals predominated in the inner plexiform layer and immunoprecipitates were also observed attached to the outer face of vesicle membranes as well as completely filling synaptic vesicles. Both clear and dense core vesicles were observed. The present results are similar to those obtained in other mammalian species showing GABA immunoreactivity in amacrine, displaced amacrine and horizontal cells

Distribution of GABA in the golden hamster retina. An immunocytochemical study.

Distribution of the inhibitory neurotransmitter GABA was studied in the golden hamster retina using immunocytochemistry at cellular and subcellular levels. GABA-immunoreactivity was observed in somata of amacrine, displaced amacrine and horizontal cells. GABA immunoreactive fibers were abundant in the inner plexiform layer. Ultrastructural analysis exhibited dense GABA-immunoreactive deposits in amacrine cell somata, processes and terminals. Immunolabelling was also observed in the cytoplasm of horizontal or interplexiform cells and displaced amacrine cells. In every case DAB deposits were observed in the cytosolic compartment, attached to the inner surface of cell membranes and to outer mitochondrial membranes. Immunolabeled terminals predominated in the inner plexiform layer and immunoprecipitates were also observed attached to the outer face of vesicle membranes as well as completely filling synaptic vesicles. Both clear and dense core vesicles were observed. The present results are similar to those obtained in other mammalian species showing GABA immunoreactivity in amacrine, displaced amacrine and horizontal cells.

Serotonergic ependymal fibres in rat and monkey: light and electron microscopic immunocytochemical study.

The distribution of serotonin (5HT) immunoreactive fibres in the ependymal epithelium of aqueductus cerebri in adult rat and monkey was studied by means of immunocytochemical methods using specific antibodies against 5HT. Light microscopic examination of immunostained coronal sections of mesencephalon showed, in tryptophan and pargylin pretreated animals, abundant serotonergic fibres distributed along the ependymal cells of the aqueduct, forming supraependymal and subependymal plexi. Serotonin fibres lying either supraependymally or ending freely in the aqueduct lumen contributed to the formation of a rich 5HT containing network on the ependymal surface. Electron microscope images showed dense 5HT-immunoreactive (5HT-IR) profiles with ultrastructural characteristics of axon terminals ending on the ependymal cells. Dense diaminobenzidine (DAB) deposits were found in the axoplasm, on outer mitochondrial surface and in vesicles. No synaptic contacts were observed between 5HT-IR terminals and ependymal cells. Unstained microvilli and cilia were also observed in the aqueduct lumen. Serotonin immunoreactivity disappeared from ependymal fibres in animals treated with parachlorophenylalanine, an inhibitor of 5HT synthesis. 5HT containing fibres described in this paper may be the source of 5HT and its metabolite 5-hydroxyindoleacetic acid (5HIAA) found in cerebrospinal fluid (CSF) and of clinical relevance in some psychiatric conditions such as depression, suicidal attempts, etc.

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.

Development of serotoninergic chick retinal neurons.

Numerous neurotransmitters have been studied in detail in the developing retina. Almost all known neurotransmitters and neuromodulators were demonstrated in vertebrate retinas using formaldehyde-induced fluorescence, uptake autoradiography or immunohistochemistry procedures. Serotoninergic (5HT) amacrine neurons were described in the inner nuclear layer (INL) of the retina with their dendrites spreading within the inner plexiform layer (IPL). The present work describes the morphological pattern of development of serotoninergic amacrine neurons with a stratified dendritic branching pattern in the chick retina from embryonic day 12 to postnatal day 7. Serotoninergic-bipolar neurons are also described. SHT-amacrine neurons have round or pear-shaped somata and primary dendritic trees oriented toward the IPL that runs through the INL, showing several varicosities. Secondary dendrites then go through the INL, without any collateral branch. At the outer and inner margin of the IPL the primary and secondary dendrites originate an outer and an inner serotoninergic network, respectively. When the primary dendritic tree reaches the IPL it deflects laterally in sublayer 1-the outer serotoninergic network. Tertiary branches then arise from the secondary dendrite and deflect in the innermost sublayer of the IPL-the inner serotoninergic network. The final pattern of branching of 5HT amacrine cells was present at embryonic day 14 and was completely developed at hatching. Serotoninergic (5HT) bipolar neurons were also present in the INL at hatching. They are weakly immunoreactive and are probably a subset of bipolar cells that accumulate serotonin from the intersynaptic cleft and are not "true" 5HT neurons.

Developmental pattern of plasminogen activator activity in chick optic lobe.

Plasminogen activators are serine proteases which play a key role in morphogenesis and tissue remodelling. Two different molecular types, tissue-type and urokinase-type, were identified and they were postulated to play a role in neural development. The developing chick optic lobe plays a central role in processing visual information. In previous studies we demonstrated the occurrence of high levels of plasminogen activator activity in this model. The aim of the present paper is to study the temporal pattern of expression of this activity and characterize the type of plasminogen activator expressed in the developing optic lobe. Using soluble fractions derived by ultracentrifugation from Triton X-100-treated membrane fractions we measured the protease activity with a radial fibrinolytic assay. Employing different inhibitors of fibrinolytic activity and a zymographic assay, we showed that the developing optic lobe expresses only one type of plasminogen activator which corresponds to an urokinase-type of 70 kDa. Our results indicate that peaks of protease activity temporally correlate with massive neuronal migration, neurite outgrowth and synapse formation and maturation. This suggests that a plasminogen activator could play a role in these developmental events. This consistent pattern of variability strongly suggests that it is developmentally regulated and, if so, it could be a reliable parameter to study neural plastic changes induced by modifications in the environmental stimulation.

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.

Immunofluorescence and glutaraldehyde fixation. A new procedure based on the Schiff-quenching method.

The immunofluorescence technique is one of the most useful methods for localizing antigens in several tissues, including the central nervous system. For immunohistochemical procedures, especially immunofluorescence methods, formaldehyde is commonly used as a fixative agent. But for some protocols, mainly in neurobiology, glutaraldehyde is necessary to recognize a number of small molecules (haptens) whose antisera have been raised using glutaraldehyde as the cross-linking agent. This is a severe limitation because glutaraldehyde gives rise to a strong autofluorescence on tissue that precludes the observation of specific immunofluorescence staining. In this paper we present a new method that allows the use of immunofluorescence techniques on glutaraldehyde-fixed tissues. The new method consists of a treatment of tissue sections with the Schiffs reagent (leucobasic fuchsin) followed by a reduction of the Schiff-dye with sodium borohydride. This reduced dye produces a quenching of glutaraldehyde-induced fluorescence on the tissue. The goal of the new method is to make possible the use of a great number of available glutaraldehyde-raised antisera for immunofluorescence techniques, a useful tool in both basic and clinical research.

Neural and astroglial effects of a chronic parachlorophenylalanine-induced serotonin synthesis inhibition.

Serotonin (5HT) is one of the classical neurotransmitters expressed earlier in the embryonic rat brain, and it was proposed as a developmental signal in the central nervous system. In the adult brain, 5HT seems to be involved in neuronal plasticity. It was postulated that S-100 protein, a glial neurotrophic factor, could be modulated by 5HT probably through the glial 5HT1A receptors. In a model of chronic inhibition of endogenous 5HT synthesis produced by the daily administration of parachlorophenylalanine (PCPA) for 2 wk, we have studied by immunohistochemical methods and digital morphometric analysis the expression of two proteins present in rat brain astrocytes: glial fibrillary acidic protein (GFAP) and S-100 protein. The effectiveness of the PCPA treatment was tested by the use of specific anti-5HT antibodies that showed absence of 5HT fibers in 5HT innervation areas like frontal cortex and hippocampus. Different effects of PCPA treatment on serotoninergic raphe nuclei were observed: dorsal raphe nucleus (DRN) seemed to be more sensitive to the PCPA's action than ventral raphe nucleus (VRN). In DRN and in the two 5HT innervation areas studied, glial cells responded to the 5HT depletion induced by PCPA showing astrocytes with large and tortuous processes. Astrocytes from 5HT-depleted regions showed higher immunostaining for S-100 protein than controls. There was not any modification in optical density of S-100 protein immunostaining in VRN, the area less sensitive to PCPA treatment. These observations indicated that astrocytes are sensitive to the 5HT level, and in presence of low 5HT concentration in the intercellular space, astrocytes could react by synthesizing glial proteins like GFAP and S-100 protein.

Unilateral enucleation induces an increase of 160 kd neurofilament in lateral geniculate nuclei synapses.

Ultrastructural synaptic changes of retinal origin in the pars dorsalis lateral geniculate nuclei (dLGN) after enucleation have been studied in this laboratory, showing a filamentous hypertrophy with maximal expression at 4-6 days post-lesion in monkeys (Pecci Saavedra et al., 1970, 1971). The aim of this work was to elucidate the nature of the newly formed filament in dLGN in post-enucleated rats. Male Wistar rats were fixed with 4% paraformaldehyde plus 0.25% glutaraldehyde in 0.1M phosphate buffer, through the abdominal aorta after 3, 5, and 7 days postenucleation. Sections obtained were incubated with antibodies to the phosphorylated portion of the 160 Kd neurofilaments (1:3000) and anti-GFAP (1:25000). There was an increase in 160 Kd neurofilament staining in axons and degenerating nerve endings in dLGN, as well as a typical astroglial immunostained reaction. Our results show that the newly formed neurofilaments after deafferentation are of the 160 Kd type, commonly present in normal axons.

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.