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R B Banati

Publications and source records attributed to R B Banati.

At least 37 records · Page 2Linked to original sources
PubMed1997

Population control of microglia: does apoptosis play a role?

Brain lesions, even of the most subtle type, are accompanied by the activation of microglia, the main immune cells of the brain. Microglial cells dramatically increase in number through proliferation and adhere to the injured neurons, where they displace the synaptic input. After proliferation, microglia gradually migrate into the nearby parenchyma and appear to decrease in number. Here we examined the possible involvement of apoptosis in the regulation of the microglial cell number using Terminal transferase mediated d-UTP Nick End-Labelling (TUNEL). In vitro, cell death is a common phenomenon in microglial cell cultures, and is enhanced by the withdrawal of the mitogen, granulocyte-macrophage colony stimulating factor. In vivo, application of the TUNEL-reaction revealed TUNEL-positive microglia beginning at day 4, with a peak 7 days after transection of the facial nerve. Surprisingly, TUNEL-labelling in vivo was localized on the outer side of the nuclear membrane and in the microglial cytoplasm, with very little staining within the nucleus itself. These TUNEL-labelled cells also lacked other classic morphological signs of apoptosis, like membrane blebbing, chromatin condensation and apoptotic bodies. These data suggest that the regulation of post-mitotic microglia is not mediated by the classic pathway of apoptosis.

Animals↗
PubMed1997

PK ('peripheral benzodiazepine')--binding sites in the CNS indicate early and discrete brain lesions: microautoradiographic detection of [3H]PK11195 binding to activated microglia.

The isoquinoline PK11195 has been suggested as a marker of glial pathology in the lesioned brain. The aim of the present study is to clarify the precise cellular location of its binding site in the central nervous system. Here, we report that in the facial nucleus after facial nerve axotomy-a lesion causing a retrograde neuronal reaction without nerve cell death while keeping the blood-brain barrier intact--activated microglia are the predominant source of lesion-induced increases of PK11195 binding. Likewise, increased PK11195 binding is seen in the gracile nucleus after anterograde neuronal injury following sciatic nerve transection. The peak of PK11195 binding, using the single isomer R-PK11195, was observed 4 days after the peripheral nerve lesion, consistent with the well-known time course of microglial activation. Photoemulsion microautoradiography confirmed the restriction of PK11195 binding to activated microglia. The increase of PK11195 binding in the facial nucleus seen after selective cell death of facial motoneurons by retrograde suicide transport of toxic ricin, a lesion that is accompanied by the rapid transformation of microglia into phagocytes, was no higher than that seen following axotomy. This suggests that the full transformation of microglia into parenchymal phagocytes is not necessary to reach maximal levels of PK11195 binding. PK11195, therefore, is a well-suited marker to detect microglial activation in areas of subtle brain pathology, where neither a disturbance of the blood-brain barrier function nor the presence of macrophages and inflammatory cells indicate an on-going disease process.

Animals↗
PubMed1996

Lack of permanent nigrostriatal dopamine deficit following 6-hydroxydopamine injection into the rat striatum. Short communication.

The lesion caused by a single 6-hydroxydopamine injection into rat striatum was evaluated. In vivo positron emission tomography using a dopamine reuptake tracer revealed no consistent reduction in striatal dopamine transporter. Amphetamine rotation test was negative up to 18 weeks. A 21% reduction in striatal dopamine seen at 11 weeks was not detectable at 18 weeks. Tyrosine hydroxylase-positive neurone counts showed no decline in substantia nigra. Our results suggest that this lesion may be subject to compensation and therefore should be used with caution in studies on neuroprotective treatments of Parkinson' disease.

Animals↗
PubMed1995

Extracellular matrix influences the biogenesis of amyloid precursor protein in microglial cells.

During axotomy studies, we discovered that the beta A4-amyloid precursor protein (APP) participates in immune responses of the central nervous system. Since microglia constitute the main immune effector cell population of this response, we used the murine microglial cell line BV-2 to analyze immune response-related APP expression. We show that interaction of microglia with the extracellular environment, particularly components of the extracellular matrix, affects APP secretion as well as intracellular APP biogenesis and catabolism. Fibronectin enhanced APP secretion and decreased the level of cellular mature transmembrane APP, whereas laminin and collagen caused a decrease in secretion and an accumulation of cellular mature APP and APP fragments. Our results demonstrate that APP plays a fundamental role in the regulation of microglial mobility, i.e. migration, initial target recognition, and binding. The decrease in APP secretion and the concomitant increase in cellular mature APP were accompanied by an accumulation of C-terminal APP fragments. Enrichment of APP and APP fragments is assumedly based on inhibition of catabolic processes that is caused by a disorganization of the actin microfilament network. These observations provide evidence that microglia, which are closely associated with certain amyloid deposits in the brain of Alzheimer patients, can play a key role in initial events of amyloidogenesis by initiating accumulation of APP and also of amyloidogenic APP fragments in response to physiological changes upon brain injury.

Alzheimer Disease↗
PubMed1995

Inflammatory reaction in experimental autoimmune encephalomyelitis (EAE) is accompanied by a microglial expression of the beta A4-amyloid precursor protein (APP).

Neuropathological studies of the amyloid depositions and senile plaques in the brains of elderly patients or patients diagnosed with Alzheimer's disease reveal the conspicuous presence of numerous proteins which are usually expressed during reactions of the immune system. This has led to speculations that the pathomechanism of neurodegenerative diseases might involve inflammatory processes. These considerations constitute the theoretical basis for therapeutic intervention with antiinflammatory drugs in neurodegenerative diseases. Here, we show that the beta A4-amyloid precursor (APP) is rapidly induced in microglia in a model of experimental autoimmune encephalomyelitis (EAE). Using specific monoclonal antibodies against APP, the first glial cells newly expressing APP immunoreactivity were found at an early preclinical stage, i.e., 24 h after T-cell transfer. At the peak of clinical disease (6 days after T-cell transfer), numerous characteristically ramified cells were strongly positive for APP. Based on morphology and double-labeling, most of the de novo APP-expressing cells were identified as microglia. Additionally, APP-immunoreactive round cells were detected in and around perivascular infiltrates. Reflecting the course of the clinical disease, the induction of APP immunoreactivity terminated in the postclinical stage, i.e., 14 days after T-cell transfer. These results support earlier work demonstrating that microglia can rapidly de novo synthesise APP not only in response to direct nerve injury (Banati et al: Glia 9:199, 1993a) but also in immune-mediated disease. Apart from its possible therapeutic relevance, such a production of APP--reminiscent of an acute phase protein-suggests a role of APP in immune and repair mechanisms of the central nervous system.

Amyloid beta-Protein Precursor↗
PubMed1995

Amyloid precursor protein (APP) expression in multiple sclerosis lesions.

The amyloid precursor protein (APP) is rapidly induced in reactive glial cells in response to several pathological stimuli including inflammation. In the present study, observations previously made in animal models of autoimmune central nervous system inflammation have been extended to the analysis of multiple sclerosis (MS) lesions. A total of thirty fresh-frozen tissue blocks from six histopathologically normal control and six MS cases have been examined immunocytochemically with monoclonal antibodies directed against either C- or N-terminal epitopes of APP. Histopathological evaluation of disease progression was based on hematoxylin-eosin and oil red O staining and immunocytochemistry for T cells, macrophages/microglia, astrocytes, and oligodendrocytes. In control cases, APP immunoreactivity was generally low and confined to blood vessel walls, oligodendrocytes in white, and neurons in grey matter. In actively demyelinating plaques, however, levels of APP immunoreactivity were high, localised on T lymphocytes, foamy macrophages, activated microglia, and reactive astrocytes including astrocytic processes. In more chronic lesions, levels of APP immunoreactivity were generally lower than in acute lesions, mainly found on reactive astrocytes, their processes and a few macrophages/microglia depending on the stage of plaque development. In addition, a few 14E-positive oligodendrocytes and, moreover, numerous axons exhibited APP immunoreactivity, which was particularly pronounced with anti-C-terminal antibodies. These results demonstrate that APP is induced on reactive glial cells but also on T lymphocytes during demyelination. The extent of APP expression appears to be correlated to histopathological lesion development and thus suggests that APP detection serves as a sensitive marker for disease progression in MS.

Adult↗
PubMed1995

Glial expression of the beta-amyloid precursor protein (APP) in global ischemia.

The beta-amyloid precursor protein (APP) bears characteristics of an acute-phase protein and therefore is likely to be involved in the glial response to brain injury. In the brain, APP is rapidly synthesized by activated glial cells in response to comparatively mild neuronal lesions, e.g., a remote peripheral nerve injury. Perfusion deficits in the brain result largely in neuronal necrosis and are a common condition in elderly patients. This neuronal necrosis is accompanied by a pronounced reaction of astrocytes and microglia, which can also be observed in animal models. We have therefore studied in the rat, immunocytochemically, the induction of APP after 30 min of global ischemia caused by four-vessel occlusion. The postischemic brain injuries were examined at survival times from 12 h to 7 days. From day 3 onward, APP immunoreactivity was strongly induced in the CA1 and CA4 regions of the rat dorsal hippocampus as well as in the dorsolateral striatum. In these areas, the majority of APP-immunoreactive cells were reactive glial fibrillary acidic protein (GFAP)-positive astrocytes, as shown by double-immunofluorescence labeling for GFAP and APP. Additionally, small ramified cells, most likely activated microglia, expressed APP immunoreactivity. In contrast, in the parietal cortex, APP immunoreactivity occurred focally in clusters of activated microglia rather than in astrocytes, as demonstrated by double-immunofluorescence labeling for APP and the microglia-binding lectin Griffonia simplicifolia isolectin B4. In conclusion, following global ischemia, APP is induced in reactive glial cells with spatial differences in the distribution pattern of APP induction in astrocytes and microglia.

Amyloid beta-Protein Precursor↗
PubMed1995

Microglial turnover in the injured CNS: activated microglia undergo delayed DNA fragmentation following peripheral nerve injury.

Microglial proliferation and activation are common events in the injured CNS. The mechanisms, however, by which activated microglia are eliminated following a pathological stimulus are still poorly understood. The present study has therefore examined microglial proliferation by 3H-thymidine autoradiography and programmed cell death by terminal transferase-mediated nick end labeling (TUNEL) and in situ end labeling (ISEL) of nuclear DNA fragments in two models of peripheral nerve injury, i.e. sciatic and hypoglossal nerve transection in the rat. In these models, microglial activation and proliferation occur in CNS projection areas, i.e. in the ventral and dorsal gray matter of lumbar spinal cord and in the nucleus gracilis after sciatic nerve transection as well as in the axotomized hypoglossal nucleus. At these sites, microglial proliferation had a relatively sharp peak between days 2 and 3 post-lesion and then rapidly declined. DNA fragmentation was detected in lectin (GSI-B4)-positive microglia from day 6 after axotomy onward, reached an apparent peak at day 21 and was downregulated by day 60, i.e. the latest time point investigated. However, the expression of bcl-2 and c-myc, i.e. genes potentially controlling programmed cell death, was found to be unchanged during this period. Programmed cell death thus appears to be one mechanism by which activated microglia are gradually eliminated following CNS injury and steady state of microglial cell numbers is achieved in vivo. Expression of microglial growth factors may be instrumental in controlling these processes.

Animals↗
PubMed1995

Reactive microglia in cerebral ischaemia: an early mediator of tissue damage?

Microglial cell activation is a rapidly occurring cellular response to cerebral ischaemia. Microglia proliferate, are recruited to the site of lesion, upregulate the expression of several surface molecules including major histocompatibility complex class I and II antigens, complement receptor and the amyloid precursor protein (APP) as well as newly expressed cytokines, e.g. interleukin-1 and transforming growth factor beta 1. The ischaemia-induced production of APP may contribute to amyloid deposition in the aged brain under conditions of hypofusion. Ultrastructurally, microglia transform into phagocytes removing necrotic neurons but still respecting the integrity of eventually surviving neurons even in the close vicinity of necrotic neurons. Microglial activation starts within a few minutes after ischaemia and thus precedes the morphologically detectable neuronal damage. It additionally involves a transient generalized response within the first 24 hours post-ischaemia even at sites without eventual neuronal cell death. In functional terms, the microglial reaction appears to be a double-edged sword in ischaemia. Activated microglia may exert a cytotoxic effector function by releasing reactive oxygen species, nitric oxide, proteinases or inflammatory cytokines. All of these cytotoxic compounds may cause bystander damage following ischaemia. Pharmacological suppression of microglial activation after ischaemia has accordingly attenuated the extent of cell death and tissue damage. However, activated microglia support tissue repair by secreting factors such as transforming growth factor beta 1 which may limit tissue damage as well as suppress astroglial scar formation. In line with ultrastructural observations microglial activation in ischaemia is a strictly controlled event. By secreting cytokines and growth factors activated microglia most likely serve seemingly opposed functions in ischaemia, i.e. maintenance as well as removal of injured neurons. Post-ischaemic pharmacological modulation of microglial intervention in the cascade of events that lead to neuronal necrosis may help to improve the structural and functional outcome following CNS ischaemia.

Animals↗
PubMed1994

Glial beta-amyloid precursor protein: expression in the dentate gyrus after entorhinal cortex lesion.

Stereotactic lesioning of the rat entorhinal cortex leads to an induction of beta-amyloid precursor protein (APP) immunoreactivity in non-neuronal cells of the deafferented dentate gyrus. Double immunofluorescence against APP and the microglia-binding isolectin B4 from Griffonia simplicifolia revealed that APP immunoreactivity was confined to activated microglia. The microglial APP expression became detectable 3 days after lesioning, reached its peak after 7 days and disappeared after 10 days. The early accumulation of APP in microglia supports the view that microglia play an important role in the initial stages of amyloid plaque formation. Such a glial APP accumulation occurs rapidly in distant but anatomically connected areas. This is in line with the preferential localization of amyloid plaques in the dentate gyrus, which is the projection field of the degenerating neurones of the entorhinal cortex in patients with developing dementia.

Amyloid beta-Protein Precursor↗
PubMed1994

Transforming growth factor beta mediates increase of mature transmembrane amyloid precursor protein in microglial cells.

By using the immortalized microglial cell line BV-2, we show that the high expression of the beta A4 amyloid precursor protein (APP), its biogenesis and metabolism is modulated by TGF beta, a cytokine with immunosuppressive activity, and by the microglia-stimulating agent LPS. TGF beta induces accumulation of cellular mature APP, the putative precursor of the amyloid subunit of Alzheimer's disease. LPS leads to an increase in cellular immature, non-amyloidogenic APP and secretion of also non-amyloidogenic APP fragments. We also demonstrate a functional involvement of ECM molecules in the regulation of microglial APP expression at mRNA and protein level by TGF beta and LPS.

Amyloid beta-Protein Precursor↗
PubMed1994

Modulation of intracellular formation of reactive oxygen intermediates in peritoneal macrophages and microglia/brain macrophages by propentofylline.

Ischemia-induced nerve cell death can partly be prevented by propentofylline, a pharmacon structurally related to xanthine derivates that interacts with the neuromodulatory function of endogenous adenosine. To evaluate a possible mechanism of neuroprotection by propentofylline, we studied its effect on the cellular production of reactive oxygen intermediates in microglial cells, which under pathological conditions can differentiate into brain macrophages, in comparison to peritoneal macrophages. Using a flow cytometric assay, we determined the intracellular formation of reactive oxygen intermediates by measuring the oxidation of the membrane-permeable and nonfluorescent dihydrorhodamine 123 to the cationic and intracellularly trapped, green fluorescent rhodamine 123 in single viable cells. Propentofylline at the therapeutic concentration of 50 microM completely inhibited the Ca(2+)-dependent Con A-induced increase in the production of reactive oxygen intermediates in peritoneal macrophages. In isolated and cultured microglial cells, which have a high spontaneous respiratory burst activity, the spontaneous production of reactive oxygen intermediates was reduced by approximately 30%. A phorbol 12-myristate 13-acetate-induced rise in the respiratory burst activity could not be inhibited by propentofylline in either cell type. An increased generation of reactive oxygen intermediates is thought to contribute to nerve cell death after brain ischemia, edema, and neurodegenerative diseases like Alzheimer's disease. These pathological conditions are all accompanied by an activation of microglial cells. We therefore suggest that the neuroprotective properties of propentofylline might in part be due to a modulation of the microglial production of potentially harmful reactive oxygen intermediates.

Animals↗
PubMed1994

Surveillance, intervention and cytotoxicity: is there a protective role of microglia?

The study of microglial cell biology has become the key to understanding the brain's fundamental tissue reactions as well as the cellular mechanisms underlying CNS disease. This article focuses on glial-neuronal interactions with special reference to human pathology. Three important areas of brain pathology are critically reviewed: multiple sclerosis and CNS inflammation, the brain in AIDS and opportunistic infections, and neurodegenerative disorders. Although microglial cytotoxicity may cause bystander damage, e.g. in ischemia, there is little evidence to support the view that microglial activation per se is pathogenic. Results suggesting that one important normal function of microglia is to protect the integrity of the central nervous system are discussed. The concept is proposed that microglia function as a highly developed guardian to the CNS.

Animals↗
PubMed1993

Cytotoxicity of microglia.

The most characteristic property of microglia is their swift activation in response to neuronal stress and their capacity for site-directed phagocytosis. The transformation of microglia into intrinsic brain macrophages appears to be under strict control and takes place if neuronal and/or terminal degeneration occurs in response to nerve lesion. The differentiation of microglia into brain macrophages is accompanied by the release of several secretory products, e.g., proteinases, cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates. Interference with the microglial activation or the productions of cytotoxic metabolites by microglia may thus offer new therapeutic opportunities for the prevention of neuronal cell death in CNS disease.

Amino Acids↗
PubMed1993

Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L.

The activation and differentiation of microglia is a prominent pathophysiological process in numerous inflammatory and demyelinating diseases of the central nervous system, including Alzheimer's disease and the AIDS encephalopathy. The tissue damage during these diseases has partly been attributed to lipid peroxidating reactive oxygen intermediates for which activated microglia are a major source. The destruction of tissue may also involve the release of proteolytic enzymes, such as the lysosomal cysteine proteinases cathepsin B and L, which are present notably in phagocytic cells. The cathepsins B and L are endopeptidases with a substrate specificity including important proteins, like myelin basic protein, extracellular matrix components, or the class II major histocompatibility complex. Because of this pathophysiological relevance the cathepsins B and L were chosen for histochemical demonstration in isolated and cultured rat microglia and measurement by a new flow cytometric method. Cathepsin B/L activity was measured flow cytometrically in single viable cells by the intracellular cleavage of non-fluorescent (Z-Phe-Arg)2-rhodamine 110 to the green fluorescent monoamide Z-Phe-Arg-rhodamine 110 and rhodamine 110. In microglia we measured a cathepsin B/L activity that was 2.5 times higher than in thioglycolate-elicited, i.e., inflammatory peritoneal rat macrophages. In elicited peritoneal macrophages the formation of fluorescent product was 6.2 times higher than in unstimulated resident peritoneal macrophages, demonstrating that the activation and differentiation of mononuclear phagocytes is accompanied by an increased cathepsin B/L enzyme activity. The subcellular localization of cathepsin B/L activity in plated viable microglia was demonstrated histochemically by the use of Z-Ala-Arg-Arg-4-methoxy-2-naphthylamide. Its blue fluorescent cleavage product 4-methoxy-2-naphthylamide was found in lysosomes. Our study shows that activated microglia are an important potential source of cathepsin B/L. This is particularly interesting as enzymatically active cathepsins have recently been found extracellularly at high levels in the senile plaques of Alzheimer's disease, which are known to contain many activated microglia. The release of proteinases by microglia may play a crucial role in the pathomechanism of tissue-destructing diseases in the brain.

Animals↗