Biomedical subjects

B Castellano

Publications and source records attributed to B Castellano.

At least 37 records · Page 2Linked to original sources

PubMed1997

Abnormal expression of the proliferating cell nuclear antigen (PCNA) in the spinal cord of the hypomyelinated Jimpy mutant mice.

In the present study, assessment of the expression of the proliferating cell nuclear antigen (PCNA), a nuclear acidic protein necessary for DNA replication that is expressed through the cell cycle, was used to investigate the proliferative capability of glial cells in the hypomyelinated Jimpy mutant mice. Spinal cords from 10-12 and 20-22 day Jimpy and normal animals were used for quantitative microscopic image analysis. Simultaneous demonstration of cycling cells and oligodendroglia, astroglia or microglia was achieved through the sequential combination of PCNA immunostaining and selective markers for these glial cells. Our results revealed that the density of PCNA-positive cells was higher in Jimpy than in normal spinal cords, this difference being more pronounced at 20-22 days than at 10-12 days and more so in white than in gray matter. In addition, Jimpy glial cells exhibited an abnormal PCNA expression, as demonstrated by quantification of the intensity of nuclear immunostaining. In comparison to normal animals, the percentage of PCNA-positive cells showing intensely stained nuclei was higher in Jimpy. About 50% of PCNA-positive cells in the Jimpy white matter were identified as cells from the oligodendrocyte line, 30% were microglial cells and 20% were astrocytes. The expression of PCNA in relation to the proliferative capability and possible cell cycle abnormalities of the different glial cell types in Jimpy is discussed.

Animals↗

PubMed1997

Development of microglia in the prenatal rat hippocampus.

The distribution and appearance of microglia cell precursors in the prenatal hippocampus were examined in embryonic day 14 (E14) to E21 rats by nucleoside diphosphatase histochemistry. For comparison, the differentiation of astroglial cells was analyzed from E17 by vimentin and glial fibrillary acidic protein immunohistochemistry. Based on morphologic features, nucleoside diphosphatase-positive microglial cell precursors were classified as ameboid microglial cells and primitive ramified microglial cells. Ameboid microglia were present in the hippocampal primordium on E14. As the hippocampus developed, however, ameboid microglia gradually transformed into primitive ramified microglia, first recognized at E19. Microglial cell precursors, often related to nucleoside diphosphatase-labeled blood vessels, were particularly observed next to the pial surface on days E14 and E17 and in the highly vascularized area around the hippocampal fissure from E19. Within the brain parenchyma, the microglial cell precursors tended to be located within the differentiating cell and neuropil layers rather than in the germinative zones. The late developing dentate gyrus remained almost devoid of microglial cell precursors before birth. Vimentin-positive astroglial processes with radial orientation were observed throughout the hippocampal subregions from E17. In contrast, glial fibrillary acidic protein-positive, radial processes were barely discernible in the fimbria and the dentate gyrus before E19. The results are discussed in relation to the possible interactive role of microglial cells in central nervous tissue development and histogenesis. Regarding the origin of hippocampal microglial cell precursors, the present observations support the view that these cells may well originate from different mesodermal sources depending on time and localization.

Animals↗

PubMed1997

Quantitative analysis of microglial reaction to a cortical excitotoxic lesion in the early postnatal brain.

This study was designed to quantify the microglial response following an injection of N-methyl-D-aspartate (NMDA) into the sensorimotor cortex of 6-day-old rats. After survival times ranging from 10 h to 28 days, cryostat sections were processed for the demonstration of microglial cells by means of tomato lectin histochemistry. The injection of NMDA caused an extensive primary lesion involving the neocortex, the rostral hippocampus, and rostral thalamus. In addition, secondary retrograde/anterograde degeneration was also observed in the ventrobasal (VB) complex of the thalamus. Microglial reactivity was already present at 10 h postlesion and restricted to areas of neuronal degeneration. Quantitative analysis was performed on digitized images using NIH Image software and a Macintosh computer. The method is based on densitometric ratios, referred to as the "reactivity grade," between the ipsilateral lesion side and the contralateral control side. Measurements were made to determine a possible increase in the number of microglial cells as well as an increase in lectin binding. The analysis showed that microglial reactivity in areas of primary degeneration peaked at 3 days postlesion, when it was significantly (P < 0.01) higher in comparison to saline-injected litter mates. Microglial response in the cerebral neocortex, showing the highest reactivity grade, as well as in other areas of primary degeneration, returned to control levels by Day 7. Microglial response in the VB complex also peaked at Day 3 (P < 0.05) but maintained this level of reactivity until 7 days postlesion (P < 0.01).

Animals↗

PubMed1997

Neonatal handling and environmental enrichment effects on emotionality, novelty/reward seeking, and age-related cognitive and hippocampal impairments: focus on the Roman rat lines.

Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rats are selected and bred for extreme divergence in two-way active avoidance acquisition. In addition, compared to RLA/Verh rats, RHA/Verh rats are (behaviorally and physiologically) less anxious or reactive to stressors, show increased novelty (sensation)-seeking behavior as well as a higher preference for rewarding substances, and are usually less efficient in learning tasks not involving shock administration. The present article reviews evidence showing that neonatal handling and/or environmental enrichment leads to enduring effects (their magnitude frequently depending upon the rat line) on those behaviors. For example, it has been found that neonatal handling reduces most of the (behavioral and physiological) signs of emotionality/anxiety in RLA/Verh rats, while environmental enrichment increases their novelty seeking (also the case with RHA/Verh rats), saccharin and ethanol intake, and sensitivity to amphetamine. Finally, initial results (currently being further elaborated upon) support a preventive action of both environmental treatments on age-related impairments in learning a spatial, water maze task as well as on hippocampal neuronal atrophy.

Animals↗

PubMed1996

Microglial response to N-methyl-D-aspartate-mediated excitotoxicity in the immature rat brain.

The intracerebral injection of N-methyl-D-aspartate (NMDA) has been proposed as a model for hypoxic-ischemic insult in the immature brain. In this light, the aim of this study was to describe the time course of the microglial reaction in the areas undergoing primary degeneration at the site of intracortical NMDA injection as well as in areas undergoing secondary anterograde and/or retrograde degeneration. Fifty nanomoles of NMDA were injected in the sensorimotor cortex of 6-day-old rats. After survival times ranging from 10 hours to 28 days, cryostat sections were stained for routine histology and for the demonstration of microglial cells by means of tomato lectin histochemistry. The areas affected by primary degeneration caused by the intracortical injection of NMDA were the neocortex, the hippocampus, and the rostral thalamus. Secondary degeneration (retrograde and anterograde) was observed in the ventrobasal complex of the thalamus. The cortical lesion also caused Wallerian degeneration of the cortical descending efferents as observed in the basilar pons. Microglial reactivity in all these areas was present at 10 hours postinjection and was restricted to the areas undergoing neuronal or axonal degeneration. Reactive microglial cells were stained intensely and showed a round or pseudopodic morphology. At 3 days, an apparent increase in the number of tomato lectin-positive cells was observed in the areas undergoing neuronal death. By 7 days after the injection, the lesion became nonprogressive, and by 14 and 28 days, microglial cells showed moderate lectin binding and a more ramified morphology.

Animals↗

PubMed1996

The microglial reaction in spinal cords of jimpy mice is related to apoptotic oligodendrocytes.

Jimpy is a shortened life-span murine mutant whose genetic disorder results in a severe hypomyelination in the central neruons system associated with a variety of glial abnormalities, including oligodendrocyte death. In this study, we report that oligodendrocyte death in jimpy occurs through an apoptotic mechanism, as demonstrated by in situ labeling of nuclear DNA fragmentation. Compared to those of normal littermates, the spinal cords of jimpy mice showed a significantly higher number of apoptotic cells. Our observations also corroborate that specific glial cell death in jimpy is restricted to oligodendrocytes, as evidenced by double labeling for DNA fragmentation and MBP immunocytochemistry. Cells labeled for DNA fragmentation were always negative for astroglial or microglial markers. Apoptotic oligodendrocytes were not aggregated into clusters and were ubiquitously distributed throughout the jimpy spinal cord, although were more numerous in white matter than in gray matter. We found no physical association between astrocytes and dying cells in jimpy. Microglial cells, however, were found closely attached to and even surrounding apoptotic cells. The possible role of microglial cells in relation to apoptotsis is discussed.

Animals↗

PubMed1996

Electrophysiological evaluation of spinal reflexes during epidural anesthesia in an experimental model.

We assessed the effects of epidural anesthesia with bupivacaine in the rat by serial recordings of spinal reflexes. The H wave from plantar muscles after electrical stimulation of the sciatic nerve evaluates a large nerve fiber spinal reflex arch. The extensor reflex response recorded from quadriceps muscle after stimulation of the contralateral tibial nerve assesses a reflex arch with small fiber afferents. After epidural injection of 0.2 mL of bupivacaine (0.25%, 0.5%, and 1.0% solutions) at the L5-L6 vertebral space, nociceptive, H, and extensor reflex responses were abolished within 1-3 min. Duration of complete blockade lasted 20-80 min, increasing with the anesthetic concentration, and complete recovery occurred after an additional period of 30-40 min. The responses recovered to amplitudes similar to preanesthesia controls, indicating that there was no damage to the nervous system. This study shows that electrophysiological recording and quantitation of nerve reflex responses is a useful and accurate method to evaluate the efficacy of local anesthetic agents.

Anesthesia, Epidural↗

PubMed1996

Reduction of the microglial cell number in rat primary glial cell cultures by exogenous addition of dibutyryl cyclic adenosine monophosphate.

The present work examined the effects induced by dibutyryl cyclic adenosine monophosphate (dB-cAMP) on microglial cells in primary glial cell cultures from newborn rats. Microglial cells were identified by OX42 immunohistochemistry and nucleoside diphosphatase histochemistry. Double staining for astrocytes was carried out by combination with glial fibrillary acidic protein immunolabeling. Addition of 0.25 mM dB-cAMP to the cultures decreased the microglial cell number about sixfold. The findings suggest that the effect of dB-cAMP on the microglial cells might be either a direct action of dB-cAMP on the microglial cells or an indirect effect mediated by the astroglial cells.

Acid Anhydride Hydrolases↗

PubMed1995

Morphology and distribution of microglial cells in the young and adult mouse cerebellum.

The morphology and distribution of microglial cells were studied in the normal cerebellum of young and adult mice using the histochemical demonstration of nucleoside diphosphatase as a specific microglial marker. Our results showed that microglial cells were present in all cerebellular lobules of both young and adult mice, but their distribution and morphology were not homogeneous throughout the cerebellum. Heterogeneity in microglial cell distribution was exclusively related to their location in the different histological layers, and no significant differences were found either between the different cerebellar lobules or between young and adult mice. Microglial density was higher in the cerebellar nuclei than in the cortex; within the cortex, the molecular layer was less densely populated by microglial cells than the granular layer and the white matter. The morphological study revealed that microglial cells were ramified in all cerebellar lobules of both young and adult mice but showed different sizes and ramification patterns as a function of their specific location in the different histological layers. Several typologies of microglial cells were described on the basis of observations in both horizontal and coronal sections. The specific layer-related pattern of microglial distribution and morphology in mouse cerebellum strongly suggests a physical and functional adaptation of these cells to the characteristics of their microenvironment.

Acid Anhydride Hydrolases↗

PubMed1995

Microglial cell reaction in the gray and white matter in spinal cords from jimpy mice. An enzyme histochemical study at the light and electron microscope level.

Jimpy is a genetic disorder which results in a severe hypomyelination in the central nervous system associated with a variety of astroglial and oligodendroglial abnormalities. In this study, we examined the morphology and distribution of microglial cells in spinal cord sections from jimpy and normal mice at 10-12 and 20-22 days postnatal using a specific microglial marker, the nucleoside diphosphatase staining. Compared to those of normal littermates, the spinal cords of jimpy mice showed an intense microglial cell reaction in white and gray matter, as revealed by quantitative analysis and light and electron microscope study. Microglial reactivity was apparent in all spinal cord areas, although it was more pronounced in white than in gray matter. The mean microglial densities in the jimpy white matter were about threefold (10-12 days) and fivefold (20-22 days) higher than in the normal, whereas in the gray matter, microglial density in jimpy was about 60% higher than in normal at both ages. Morphologically, microglial cells in the normal spinal cord showed a ramified appearance, similar in size and ramification pattern to those reported in other normal CNS areas. In contrast, microglial cells in the jimpy spinal cord showed a reactive morphology, characterized by a shortening and coarsening of their cell processes, swelling of their cell body and accumulation of lipid inclusions. Reactive microglial cells were found in close association with axons and oligodendroglial cells. The possible role of microglial cells in hypomyelination is discussed.

Acid Anhydride Hydrolases↗

PubMed1994

Transitory disappearance of microglia during the regeneration of the lizard medial cortex.

In normal lizards, microglial cells populate the medial cortex (a zone homologous to the hippocampal fascia dentata), with a preferential distribution along the border between the granular cell layer and the plexiform layers. Intraperitoneal injection of the neurotoxin 3-acetylpyridine (3AP) induces a selective lesion in the medial cortex with a rapid degeneration of the granular layer and its zinc-enriched axonal projection. Within 6-8 weeks, the granular layer is, however, repopulated by a new set of neurons generated in the subjacent ependyma and the cell debris is removed. The aim of this study was to determine to what extent microglia were involved in the scavenging processes during the regeneration process. To this end we studied the brains of regenerating lizards at different times after 3AP lesion, visualising microglial cells by the nucleoside diphosphatase (NDPase) histochemical reaction. Surprisingly, we found that stained microglial cells disappeared 6-8 hours after 3AP injection and remained absent until 10-15 days after injection. One month postlesion an increased population of microglial cells was found scattered throughout all plexiform layers of the cortex. Thorough examination of semithin and ultrathin sections confirmed the absence of microglia in the medial cortex of recent lesioned animals but the presence of an exuberant population after 1 month postlesion. In the tissue, phagocytotic scavenging was carried out by radial ependymocytes, not by microglia.

Acid Anhydride Hydrolases↗

PubMed1994

Microglial and astroglial reactions to anterograde axonal degeneration: a histochemical and immunocytochemical study of the adult rat fascia dentata after entorhinal perforant path lesions.

The reaction of microglial and a stroglial cells to anterograde axonal degeneration was studied in the fascia dentata of adult rats at various timepoints after removal of the entorhinal perforant path projection. Microglial cells were identified by histochemical staining for nucleoside diphosphatase (NDPase) at light and electron microscopical levels. Astroglial cells were stained immunocytochemically for glial fibrillary acidic protein (GFAP). Activated astroglial cells and some microglial cells also stained immunocytochemically for the intermediate filament protein vimentin. Phagocytotic activity was detected by histochemical staining for acid phosphatase. The postlesional connective reorganization of the cholinergic septohippocampal projection was monitored by histochemical staining for acetylcholinesterase. Twenty-four hours after entorhinal cortex ablation, microglial cells in the perforant path zones of the fascia dentata and the adjacent neuropil reacted by shortening and coarsening of processes and an increase in NDPase reactivity. These changes occurred prior to a noticeable increase in GFAP immunoreactivity and hypertrophy of astroglial cells (first evident on postlesional day 2) or sprouting of cholinergic septohippocampal fibres (first evident on day 3). There was evidence of an early, local proliferation of microglial cells in the denervated perforant path zones and migration into these zones of microglial cells from adjacent intact areas. The specific accumulation of strongly stained microglial cells within the denervated parts of the dentate molecular layer persisted for at least 4 weeks, while the astroglial reaction subsided at 3 weeks. The results demonstrate an early activation of microglial cells by axonal degeneration, and indicate that these cells may play a pivotal, inductive role in the subsequent glial and neural events.

5'-Nucleotidase↗

PubMed1994

Demonstration of poly-N-acetyl lactosamine residues in ameboid and ramified microglial cells in rat brain by tomato lectin binding.

This study was designed to demonstrate the localization of poly-N-acetyl lactosamine residues in postnatal and adult rat brain, visualized by their specific binding to a lectin obtained from Lycopersicon esculentum (tomato). Lectin histochemistry was carried out on cryostat, paraffin, and vibratome sections and was examined by light microscopy. Selected vibratome sections were processed for electron microscopy. Our results showed that tomato lectin histochemistry was found in relation to blood vessels and glial cells in both postnatal and adult rat brain. Since tomato lectin-positive glial cells did not show GFAP immunoreactivity and displayed the same morphological features and overall distribution as nucleoside diphosphatase (NDPase)-positive cells, they were consequently identified as microglial cells. At the electron microscopic level, both ameboid and ramified microglial cells displayed intracytoplasmic and plasma membrane lectin reactivity. In postnatal brain, ameboid microglial cells always showed stronger binding of tomato lectin compared with ramified microglial cells in the adult brain. The putative significance of this decrease in poly-N-acetyl lactosamine from ameboid to ramified microglial cells and the possible role(s) of this sugar residue are discussed.

Acid Anhydride Hydrolases↗

PubMed1993

Microglial and astroglial reactions to ischemic and kainic acid-induced lesions of the adult rat hippocampus.

The aim of this study was to characterize the microglial and astroglial reactions to degeneration of (a) hippocampal CA1 pyramidal cells and dentate hilar neurons induced by cerebral ischemia and (b) CA3 pyramidal cells and dentate hilar neurons induced by intraventricular injections of kainic acid (KA). The microglial reactions to ischemia, as monitored by histochemical staining for the enzyme nucleoside diphosphatase (NDPase) and immunohistochemical staining for the complement type 3 receptor (CR3), could be divided into (1) initial and generalized, but transient, reactions which also included areas devoid of subsequent neural degeneration and (2) protracted, degeneration-specific reactions in the areas with neural degeneration. Due to more widespread hippocampal involvement a similar distinction was not possible after KA lesions. After both ischemia and KA application the protracted degeneration-specific reactions were characterized by increased NDPase/CR3 reactivity and prominent morphological changes. In the dentate hilus, reactive microglial cells clustered around the degenerating hilar neurons. In stratum radiatum of CA1, reactive microglial cells transformed into either (1) "rod cells," aligned along the postischemic, degenerating pyramidal cell dendrites, followed by subsequent transformation into ameboid-like cells, or (2) "bushy" cells, in response to degeneration of Schaffer collaterals induced by KA lesioning of CA3 pyramidal cells. Within stratum radiatum of the KA-lesioned CA3, where both dendrites and axons were degenerating, the microglial cells developed into stellate cells with thickened, retracted processes and plump cell bodies. These cells were supplemented by rounded macrophage-like cells. Astroglial reactions, monitored by immunohistochemical staining for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin (VIM), and the normal plasma constituent immunoglobulin G (IgG), showed an initial and generalized astroglial immunoreactivity for IgG, which paralleled the initial and transient microglial reactions, while the reactive changes in GFAP and VIM immunohistochemistry paralleled the protracted, degeneration-specific reactions with regard to timing, strength, and distribution. In the KA-lesioned CA3, the most prominent finding was a prompt loss of astroglial GFAP immunoreactivity corresponding to the degenerating pyramidal cell layer and the adjacent mossy fiber layer. The results strongly indicate that stimuli other than neural degeneration initiated the activation of both microglial and astroglial cells, which then upon further activation by actual neuronal damage and degeneration adjust according to which neuronal structures were undergoing degeneration.

Acid Anhydride Hydrolases↗

PubMed1991

Identification and distribution of microglial cells in the cerebral cortex of the lizard: a histochemical study.

The histochemical demonstration of nucleoside diphosphatase as a specific microglial marker was used to study the distribution of this glial cell type in the cerebral cortex of Podarcis muralis and Podarcis hispanica. Our results showed that in both species, NDPase staining was specific for the microglial cell population and that microglial cells displayed a specific localization pattern in the different cortical areas. In the medal cortex, microglial cells were principally found in the outer and inner plexiform layers in the strata adjacent to the granular layer. Moreover, some microglial cells were found near the ependymal layer, but no microglial cells were normally present near the brain surface and never in the deep inner plexiform layer. In the dorsomedial cortex, microglial cells were found near the brain surface in the outer plexiform layer, in the upper part of the granular layer, and near the ependymal layer. No microglial cells were found, however, in the outer and inner plexiform layers adjacent to the granular layer. Finally, in the dorsolateral cortex, microglial cells were located in the upper part of the outer plexiform layer, in and bordering the granular layer, and scattered in the inner plexiform layer. This layered-pattern distribution of microglial cell population in the cerebral cortex of the lizard differs from the apparently homogeneous distribution of microglia in the brain of mammals.

Acid Anhydride Hydrolases↗

PubMed1991

Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study.

Neural mouse xenografts undergoing rejection in the adult recipient rat brain were characterized with regard to infiltrating host leukocytes and reactions of graft and host astro- and microglial cells. Rejection occurred within 35 days with infiltration of the grafts by in particular macrophages and T-cells as well as blood-brain barrier (BBB) leakage for IgG. In the surrounding host brain microglial cells showed increased histochemical staining for nucleoside diphosphatase (NDPase) and increased immunocytochemical expression of complement receptor type 3 (CR3), while astroglial cells displayed an increased immunoreactivity for glial fibrillary acidic protein (GFAP). Light microscopic findings of rat major histocompatibility complex (MHC) antigen class I on microglial cells, endothelial cells and leukocytes were confirmed at the ultrastructural level and extended to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglia. We suggest that host macrophages and activated host and xenograft microglial cells act in situ as immunostimulatory cells on T-helper cells, and that increased levels of donor MHC antigen class I may further enhance the killer activity exerted by host T-cytotoxic cells.

Acid Anhydride Hydrolases↗

PubMed1991

A double staining technique for simultaneous demonstration of astrocytes and microglia in brain sections and astroglial cell cultures.

We developed a double staining technique for simultaneous demonstration of astrocytes and microglial cells in histological brain sections and cell cultures. The procedure included a histochemical stain specific for microglial cells and an immunocytochemical stain specific for astroglial cells, with postponement of the final visualization of the staining products until both reactions had been performed. First, microglial cells were specifically but invisibly labeled by histochemical reaction for nucleoside diphosphatase (NDPase). Then the astroglial cells were labeled by performing the first parts of the immunocytochemical reaction for glial fibrillary acidic protein (GFAP). Finally, in a series of intervening steps, the NDPase reaction product was visualized and stabilized by treatment with ammonium sulfide and silver nitrate, while the 1-naphthol basic dye method was used to visualize the GFAP immunoreactive product. As an end product, the NDPase-positive microglial cells were brown and the GFAP-reactive astroglial cells blue. The two types of glial cells were clearly distinguishable in vibratome sections of rat brain tissue and in primary astroglial cell cultures, and we never observed cells that stained for both NDPase and GFAP. When the GFAP antibody was replaced by the OX-42 antibody, which recognizes microglial cells and macrophages, double staining of microglial cells was observed. The staining protocol has wide applications in studies of the functional interactions between microglial and astroglial cells in the normal brain and in different pathological states with neuronal or axonal degeneration, just as it can be used for experimental studies in cell cultures.

Acid Anhydride Hydrolases↗

PubMed1990

Histochemical demonstration of purine nucleoside phosphorylase (PNPase) in microglial and astroglial cells of adult rat brain.

The histochemical localization of enzymes associated with purine nucleoside metabolism indicates that glial cells might participate in the regulation of these compounds in the central nervous system. In the present study we examined the histochemical localization of purine nucleoside phosphorylase (PNPase) in sections from adult rat brain. Some sections were also sequentially stained immunocytochemically for astroglial or microglial cells utilizing glial fibrillary acidic protein (GFAP) or OX-42 antibodies, respectively. Our observations showed that PNPase was restricted to glial cells, whereas neurons always remained negative. Brain sections stained for both PNPase and GFAP showed that the GFAP-positive astroglial cells were always PNPase positive. Other PNPase-positive but GFAP-negative cells were also observed. These cells resembled microglial cells, and brain sections reacted for both PNPase and OX-42 confirmed this by showing that the major part of OX-42-positive microglial cells were PNPase positive. In these sections, the PNPase-positive but OX-42-negative cells present resembled astroglial cells. From our double staining experiments, we conclude that PNPase is present in both astroglial and microglial cells in normal adult brain.

Animals↗