Josep Saura

High-yield isolation of murine Microglia by mild trypsinization

Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05–0.12%) in the presence of 0.2–0.5 mM EDTA and 0.5–0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as > 98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony‐stimulating factor and lipopolysaccharide, alone or in the presence of interferon γ, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor‐κB translocation, NO, and tumor necrosis α release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals.

Increased monoamine oxidase b activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography

The aetiology and pathogenesis of Alzheimers disease are currently poorly understood, but symptomatic disease is associated with amyloid plaques, neurofibrillary tangles, neuronal loss and numerous alterations of neurotransmitter systems in the CNS. Monoamine oxidase type B is known to be increased in Alzheimer diseased brains. The distribution and abundance of catalytic sites for monoamine oxidases A and B in post mortem human brains of 11 Alzheimer disease cases and five age-matched controls were investigated by quantitative enzyme radioautography. Using tritiated monoamine oxidase inhibitors (Ro41-1049 and lazabemide)--as high affinity substrates selective for monoamine oxidases A and B, respectively--it was found that monoamine oxidase B activity increased up to three-fold exclusively in temporal, parietal and frontal cortices of Alzheimer disease cases compared with controls. This increase was restricted to discrete patches (approximately 185 microns in diameter) which occupied approximately 12% of the cortical areas examined. In other brain regions (hippocampal formation >> caudate-putamen > cerebellum), patches of [3H]lazabemide-enriched binding were less abundant. [3H]Ro41-1049 binding (i.e. monoamine oxidase A) was unchanged in all tissues of diseased versus control brains. The monoamine oxidase B-enriched patches in all cortical regions correlated, in their distribution and frequency, with glial fibrillary acidic protein-immunoreactive clusters of astrocytes. Diffuse and mature beta-amyloid-immunoreactive senile plaques as well as patches of high density binding of [3H]PK-11195--a high-affinity ligand for peripheral-type (mitochondrial) benzodiazepine binding sites in microglia/macrophages--were found throughout Alzheimer diseased cortices. The up-regulation of monoamine oxidase B in plaque-associated astrocytes in Alzheimers disease--in analogy to its proposed role in neurodegenerative disorders such as Parkinsons disease--might, indirectly, be a potential source of cytotoxic free radicals. Lazabemide, a selective reversible monoamine oxidase B inhibitor, is currently under clinical evaluation for the treatment of Parkinsons and Alzheimers diseases. We conclude that enzyme radioautography with [3H]lazabemide is a reliable high resolution assay for plaque-associated astroglioses in Alzheimers disease. Its clinical diagnostic utility for positron emission tomography or single photon emission computer tomography studies is being investigated.

Microglial cells in astroglial cultures: a cautionary note

Primary rodent astroglial-enriched cultures are the most popular model to study astroglial biology in vitro. From the original methods described in the 1970s a great number of minor modifications have been incorporated into these protocols by different laboratories. These protocols result in cultures in which the astrocyte is the predominant cell type, but astrocytes are never 100% of cells in these preparations. The aim of this review is to bring attention to the presence of microglia in astroglial cultures because, in my opinion, the proportion of and the role that microglial cells play in astroglial cultures are often underestimated. The main problem with ignoring microglia in these cultures is that relatively minor amounts of microglia can be responsible for effects observed on cultures in which the astrocyte is the most abundant cell type. If the relative contributions of astrocytes and microglia are not properly assessed an observed effect can be erroneously attributed to the astrocytes. In order to illustrate this point the case of NO production in activated astroglial-enriched cultures is examined. Lipopolysaccharide (LPS) induces nitric oxide (NO) production in astroglial-enriched cultures and this effect is very often attributed to astrocytes. However, a careful review of the published data suggests that LPS-induced NO production in rodent astroglial-enriched cultures is likely to be mainly microglial in origin. This review considers cell culture protocol factors that can affect the proportion of microglial cells in astroglial cultures, strategies to minimize the proportion of microglia in these cultures, and specific markers that allow the determination of such microglial proportions.

Neuroanatomical Abnormalities in Behaviorally Characterized APPV717F Transgenic Mice

Histological analyses were performed on the brains of APP(V717F) transgenic (Tg)mice previously studied in a battery of behavioral tests. We describe here the regional and age-dependent deposition of amyloid in both heterozygous and homozygous Tg mice. We also report that Tg mice show significant and age-dependent changes in synaptic density measured by synaptophysin immunoreactivity. Surprisingly, a rather marked hippocampal atrophy is observed as early as 3 months of age in Tg mice (20-40%). Statistical analyses revealed that the deficits in object recognition memory are related to the number of amyloid deposits in specific brain regions, whereas deficits in spatial reference and working memory are related to the changes in synaptic density and hippocampal atrophy. Our study suggests that the behavioral deficits observed in Tg mice are only in part related to amyloid deposition, but are also related to neuroanatomical alterations secondary to overexpression of the APP(V717F) transgene and independent of amyloid deposition.

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinsons and Alzheimers diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide‐induced NO release and NO synthase‐II expression in a time‐ and concentration‐dependent manner. CGS21680 potentiation of lipopolysaccharide‐induced NO release was suppressed by the A2A receptor antagonist ZM‐241385 and did not occur on mixed glial cultures from A2A receptor‐deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase‐II inhibitor 1400W abolished NO production, and NO synthase‐II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase‐II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.

Differential age-related changes of mao-a and mao-b in mouse brain and pe peripheral organs

Distribution and age-related changes of MAO in BL/C57 mouse were studied by quantitative enzyme radioautography with [3H]Ro41-1049 and [3H]Ro19-6327. In the brain, MAO-A was highest in locus coeruleus and interpeduncular nucleus, and MAO-B in raphe nuclei, paraventricular thalamic nucleus, and ependyma of ventricles. Extremely high MAO-B levels were also measured in the choroid plexus in contrast to the very low MAO-B levels in rat choroid plexus. With aging, brain MAO-A showed a clear decrease between 4 and 9 weeks, followed by no change between 9 weeks and 19 months, and a slight increase between 19 and 25 months. On the other hand, all brain structures showed age-related increases in MAO-B. Peripheral organs showed different patterns of MAO age-related changes. Particularly interesting was the marked MAO-B increase in heart, parallel to the MAO-A increase in rat heart. Also of interest is the decrease of liver MAO-B in old animals, which, together with the increase of MAO-B in the brain, might underlie the high sensitivity of old BL/C57 mice to MPTP.

Biphasic and Region-Specific MAO-B Response to Aging in Normal Human Brain

Variations of monoamine oxidases (MAO) A and B were studied during aging in 27 human subjects (age range 17-93 years) in 18 brain structures of temporal cortex, frontal gyrus, hippocampal formation, striatum, cerebellum, and brainstem. [3H]Ro41-1049 and [3H]lazabemide were used as selective radioligands to image and quantify MAO-A and MAO-B respectively by enzyme autoradiography. Postmortem delay or time of tissue storage did not affect MAO-A or MAO-B levels. There was, moreover, no evidence of sexual dimorphism. A marked age-related increase in MAO-B was observed in most structures. This increase started at the age of 50-60 years. Before this age, MAO-B levels were constant in all structures studied. MAO-B-rich senile plaques were observed in some cortical areas but they did not significantly influence the age-related MAO-B increase. Surprisingly, no age-related MAO-B changes were observed in the substantia nigra. In contrast to MAO-B, no clear age-related changes in MAO-A were observed, indicating an independent regulation of the two isoenzymes, also suggested by the cross-correlation analysis of these data.

Localization of monoamine oxidases in human peripheral tissues

Localization of monoamine oxidases (MAO) A and B and beta-adrenoceptors, was studied in aged human peripheral tissues (age 68-80 years) by quantitative autoradiography. The tissues analyzed were heart, lung, liver, kidney, spleen and duodenum. [3H]Ro41-1049 and [3H]lazabemide, two recently characterized selective radioligands were used to map MAO-A and MAO-B respectively. The regional pattern of distribution of MAO-A and MAO-B did not differ markedly, except in kidney and especially in duodenum. Highest levels of MAOs were measured in liver, and lowest in spleen. MAO-A was more abundant than MAO-B in lung and duodenal mucosa, and the reverse was true in myocardium. These results show marked differences in the abundance and patterns of distribution of MAOs, particularly MAO-B, in human and rodent peripheral tissues.

Upregulation of CCAAT/enhancer binding protein β in activated astrocytes and microglia

The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPβ in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPβ expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPβ in untreated mixed glial cultures. C/EBPβ is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS‐induced upregulation of glial C/EBPβ is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPβ nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS‐induced increase in microglial C/EBPβ is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPβ as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPβ expression in activated glial cells in vivo. These findings support a role for C/EBPβ in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPβ‐regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation.