Visualization of incrementally learned projection trajectories for longitudinal data.

Longitudinal studies that continuously generate data enable the capture of temporal variations in experimentally observed parameters, facilitating the interpretation of results in a time-aware manner. We propose IL-VIS (incrementally learned visualizer), a new machine learning pipeline that incrementally learns and visualizes a progression trajectory representing the longitudinal changes in longitudinal studies. At each sampling time point in an experiment, IL-VIS generates a snapshot of the longitudinal process on the data observed thus far, a new feature that is beyond the reach of classical static models. We first verify the utility and correctness of IL-VIS using simulated data, for which the true progression trajectories are known. We find that it accurately captures and visualizes the trends and (dis)similarities between high-dimensional progression trajectories. We then apply IL-VIS to longitudinal multi-electrode array data from brain cortical organoids when exposed to different levels of quinolinic acid, a metabolite contributing to many neuroinflammatory diseases including Alzheimer's disease, and its blocking antibody. We uncover valuable insights into the organoids' electrophysiological maturation and response patterns over time under these conditions.

A Review of the Evidence for Tryptophan and the Kynurenine Pathway as a Regulator of Stem Cell Niches in Health and Disease.

Stem cells are ubiquitously found in various tissues and organs in the body, and underpin the body's ability to repair itself following injury or disease initiation, though repair can sometimes be compromised. Understanding how stem cells are produced, and functional signaling systems between different niches is critical to understanding the potential use of stem cells in regenerative medicine. In this context, this review considers kynurenine pathway (KP) metabolism in multipotent adult progenitor cells, embryonic, haematopoietic, neural, cancer, cardiac and induced pluripotent stem cells, endothelial progenitor cells, and mesenchymal stromal cells. The KP is the major enzymatic pathway for sequentially catabolising the essential amino acid tryptophan (TRP), resulting in key metabolites including kynurenine, kynurenic acid, and quinolinic acid (QUIN). QUIN metabolism transitions into the adjoining de novo pathway for nicotinamide adenine dinucleotide (NAD) production, a critical cofactor in many fundamental cellular biochemical pathways. How stem cells uptake and utilise TRP varies between different species and stem cell types, because of their expression of transporters and responses to inflammatory cytokines. Several KP metabolites are physiologically active, with either beneficial or detrimental outcomes, and evidence of this is presented relating to several stem cell types, which is important as they may exert a significant impact on surrounding differentiated cells, particularly if they metabolise or secrete metabolites differently. Interferon-gamma (IFN-γ) in mesenchymal stromal cells, for instance, highly upregulates rate-limiting enzyme indoleamine-2,3-dioxygenase (IDO-1), initiating TRP depletion and production of metabolites including kynurenine/kynurenic acid, known agonists of the Aryl hydrocarbon receptor (AhR) transcription factor. AhR transcriptionally regulates an immunosuppressive phenotype, making them attractive for regenerative therapy. We also draw attention to important gaps in knowledge for future studies, which will underpin future application for stem cell-based cellular therapies or optimising drugs which can modulate the KP in innate stem cell populations, for disease treatment.

Perivascular macrophages create an intravascular niche for CD8+ T cell localisation prior to the onset of fatal experimental cerebral malaria.

OBJECTIVES: The immunologic events that build up to the fatal neurological stage of experimental cerebral malaria (ECM) are incompletely understood. Here, we dissect immune cell behaviour occurring in the central nervous system (CNS) when Plasmodium berghei ANKA (PbA)-infected mice show only minor clinical signs. METHODS: A 2-photon intravital microscopy (2P-IVM) brain imaging model was used to study the spatiotemporal context of early immunological events in situ during ECM. RESULTS: Early in the disease course, antigen-specific CD8+ T cells came in contact and arrested on the endothelium of post-capillary venules. CD8+ T cells typically adhered adjacent to, or were in the near vicinity of, perivascular macrophages (PVMs) that line post-capillary venules. Closer examination revealed that CD8+ T cells crawled along the inner vessel wall towards PVMs that lay on the abluminal side of large post-capillary venules. 'Activity hotspots' in large post-capillary venules were characterised by T-cell localisation, activated morphology and clustering of PVM, increased abutting of post-capillary venules by PVM and augmented monocyte accumulation. In the later stages of infection, when mice exhibited neurological signs, intravascular CD8+ T cells increased in number and changed their behaviour, actively crawling along the endothelium and displaying frequent, short-term interactions with the inner vessel wall at hotspots. CONCLUSION: Our study suggests an active interaction between PVM and CD8+ T cells occurs across the blood-brain barrier (BBB) in early ECM, which may be the initiating event in the inflammatory cascade leading to BBB alteration and neuropathology.

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

P2X7 receptor signaling during adult hippocampal neurogenesis.

Neurogenesis is a persistent and essential feature of the adult mammalian hippocampus. Granular neurons generated from resident pools of stem or progenitor cells provide a mechanism for the formation and consolidation of new memories. Regulation of hippocampal neurogenesis is complex and multifaceted, and numerous signaling pathways converge to modulate cell proliferation, apoptosis, and clearance of cellular debris, as well as synaptic integration of newborn immature neurons. The expression of functional P2X7 receptors in the central nervous system has attracted much interest and the regulatory role of this purinergic receptor during adult neurogenesis has only recently begun to be explored. P2X7 receptors are exceptionally versatile: in their canonical role they act as adenosine triphosphate-gated calcium channels and facilitate calcium-signaling cascades exerting control over the cell via calcium-encoded sensory proteins and transcription factor activation. P2X7 also mediates transmembrane pore formation to regulate cytokine release and facilitate extracellular communication, and when persistently stimulated by high extracellular adenosine triphosphate levels large P2X7 pores form, which induce apoptotic cell death through cytosolic ion dysregulation. Lastly, as a scavenger receptor P2X7 directly facilitates phagocytosis of the cellular debris that arises during neurogenesis, as well as during some disease states. Understanding how P2X7 receptors regulate the physiology of stem and progenitor cells in the adult hippocampus is an important step towards developing useful therapeutic models for regenerative medicine. This review considers the relevant aspects of adult hippocampal neurogenesis and explores how P2X7 receptor activity may influence the molecular physiology of the hippocampus, and neural stem and progenitor cells.

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells.

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

HIV brain latency as measured by CSF BcL11b relates to disrupted brain cellular energy in virally suppressed HIV infection.

OBJECTIVE: We investigated whether HIV brain latency was associated with brain injury in virally suppressed HIV infection. DESIGN: Observational cross-sectional and longitudinal study. METHODS: The study included 26 virally suppressed HIV-infected men (61.5% with HIV-associated neurocognitive disorder) who undertook cerebrospinal fluid (CSF) analyses at baseline. They also completed a proton magnetic resonance spectroscopy (1H MRS) and neuropsychological assessments at baseline and 18 months. To quantify whether there was residual brain HIV transcription, we measured CSF HIV-tat. As an HIV brain latency biomarker, we used concentrations of CSF BcL11b - a microglia transcription factor that inhibits HIV transcription. Concurrently, we assessed neuroinflammation with CSF neopterin, neuronal injury with CSF neurofilament light-chain (NFL), and in-vivo neurochemistry with 1H MRS of N-acetyl aspartate (NAA), choline (Cho), creatine, myo-inositol (MI), glutamine/glutamate (Glx) in the frontal white matter (FWM), posterior cingulate cortex (PCC), and caudate nucleus area. RESULTS: Baseline adjusted regression models for neopterin, NFL, and tat showed that a higher CSF BcL11b was consistently associated with lower FWM creatine (when adjusted for neopterin: ß = -0.30, P = 0.15; when adjusted for NFL: ß = -0.47, P = 0.04; and when adjusted for tat: ß = -0.47, P = 0.02). In longitudinal analyses, we found no time effect, but a consistent BcL11b altering effect on FWM creatine. The effect reached a significant moderate effect size range when corrected for CSF NFL (ß = -0.36, P = 0.02) and CSF tat (ß = -0.34, P = 0.02). CONCLUSIONS: Reduced frontal white matter total creatine may indicate subclinical HIV brain latency-related injury. H MRS may offer a noninvasive option to measure HIV brain latency.

P2X7 Receptors Regulate Phagocytosis and Proliferation in Adult Hippocampal and SVZ Neural Progenitor Cells: Implications for Inflammation in Neurogenesis.

Identifying the signaling mechanisms that regulate adult neurogenesis is essential to understanding how the brain may respond to neuro-inflammatory events. P2X7 receptors can regulate pro-inflammatory responses, and in addition to their role as cation channels they can trigger cell death and mediate phagocytosis. How P2X7 receptors may regulate adult neurogenesis is currently unclear. Here, neural progenitor cells (NPCs) derived from adult murine hippocampal subgranular (SGZ) and cerebral subventricular (SVZ) zones were utilized to characterize the roles of P2X7 in adult neurogenesis, and assess the effects of high extracellular ATP, characteristic of inflammation, on NPCs. Immunocytochemistry found NPCs in vivo and in vitro expressed P2X7, and the activity of P2X7 in culture was demonstrated using calcium influx and pore formation assays. Live cell and confocal microscopy, in conjunction with flow cytometry, revealed P2X7+ NPCs were able to phagocytose fluorescent beads, and this was inhibited by ATP, indicative of P2X7 involvement. Furthermore, P2X7 receptors were activated with ATP or BzATP, and 5-ethynyl-2'-deoxyuridine (EdU) used to observe a dose-dependent decrease in NPC proliferation. A role for P2X7 in decreased NPC proliferation was confirmed using chemical inhibition and NPCs from P2X7-/- mice. Together, these data present three distinct roles for P2X7 during adult neurogenesis, depending on extracellular ATP concentrations: (a) P2X7 receptors can form transmembrane pores leading to cell death, (b) P2X7 receptors can regulate rates of proliferation, likely via calcium signaling, and (c) P2X7 can function as scavenger receptors in the absence of ATP, allowing NPCs to phagocytose apoptotic NPCs during neurogenesis. Stem Cells 2018;36:1764-1777.

Bcl11b-A Critical Neurodevelopmental Transcription Factor-Roles in Health and Disease.

B cell leukemia 11b (Bcl11b) is a zinc finger protein transcription factor with a multiplicity of functions. It works as both a genetic suppressor and activator, acting directly, attaching to promoter regions, as well as indirectly, attaching to promoter-bound transcription factors. Bcl11b is a fundamental transcription factor in fetal development, with important roles for the differentiation and development of various neuronal subtypes in the central nervous system (CNS). It has been used as a specific marker of layer V subcerebral projection neurons as well as striatal interneurons. Bcl11b also has critical developmental functions in the immune, integumentary and cardiac systems, to the extent that Bcl11b knockout mice are incompatible with extra-uterine life. Bcl11b has been implicated in a number of disease states including Huntington's disease, Alzheimer's disease, HIV and T-Cell malignancy, amongst others. Bcl11b is a fascinating protein whose critical roles in the CNS and other parts of the body are yet to be fully explicated. This review summarizes the current literature on Bcl11b and its functions in development, health, and disease as well as future directions for research.

The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells.

RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.

Recent evidence for an expanded role of the kynurenine pathway of tryptophan metabolism in neurological diseases.

The kynurenine pathway (KP) of tryptophan metabolism has emerged in recent years as a key regulator of the production of both neuroprotective (e.g. kynurenic and picolinic acid, and the essential cofactor NAD+) and neurotoxic metabolites (e.g. quinolinic acid, 3-hydroxykynurenine). The balance between the production of the two types of metabolites is controlled by key rate-limiting enzymes such as indoleamine-2,3-dioxygenase (IDO-1), and in turn, molecular signals such as interferon-γ (IFN-γ), which activate the KP metabolism of tryptophan by this enzyme, as opposed to alternative pathways for serotonin and melatonin production. Dysregulated KP metabolism has been strongly associated with neurological diseases in recent years, and is the subject of increasing efforts to understand how the metabolites are causative of disease pathology. Concurrent with these endeavours are drug development initiatives to use inhibitors to block certain enzymes in the pathway, resulting in reduced levels of neurotoxic metabolites (e.g. quinolinic acid, an excitotoxin and N-Methyl-d-Aspartate (NMDA) receptor agonist), while in turn enhancing the bioavailability of the neuroprotective metabolites such as kynurenic acid. Neurodegenerative diseases often have a substantial autoimmune or inflammatory component; hence a greater understanding of how KP metabolites influence the inflammatory cascade is required. Additionally, challenges exist in diseases like multiple sclerosis (MS) and motor neurone disease (MND), which do not have reliable biomarkers. Clinical diagnosis can often be prolonged in order to exclude other diseases, and often diagnosis occurs at an advanced state of disease pathology, which does not allow a lengthy time for patient assessment and intervention therapies. This review considers the current evidence for involvement of the KP in several neurological diseases, in biomarkers of disease and also the parallels that exist in KP metabolism with what is known in other diseases such as HIV, Alzheimer's disease/dementia, infection, immune privilege and cardiovascular disease. This article is part of the Special Issue entitled 'The Kynurenine Pathway in Health and Disease'.

Current Evidence for a Role of the Kynurenine Pathway of Tryptophan Metabolism in Multiple Sclerosis.

The kynurenine pathway (KP) is the major metabolic pathway of the essential amino acid tryptophan (TRP). Stimulation by inflammatory molecules, such as interferon-γ (IFN-γ), is the trigger for induction of the KP, driving a complex cascade of production of both neuroprotective and neurotoxic metabolites, and in turn, regulation of the immune response and responses of brain cells to the KP metabolites. Consequently, substantial evidence has accumulated over the past couple of decades that dysregulation of the KP and the production of neurotoxic metabolites are associated with many neuroinflammatory and neurodegenerative diseases, including Parkinson's disease, AIDS-related dementia, motor neurone disease, schizophrenia, Huntington's disease, and brain cancers. In the past decade, evidence of the link between the KP and multiple sclerosis (MS) has rapidly grown and has implicated the KP in MS pathogenesis. KP enzymes, indoleamine 2,3-dioxygenase (IDO-1) and tryptophan dioxygenase (highest expression in hepatic cells), are the principal enzymes triggering activation of the KP to produce kynurenine from TRP. This is in preference to other routes such as serotonin and melatonin production. In neurological disease, degradation of the blood-brain barrier, even if transient, allows the entry of blood monocytes into the brain parenchyma. Similar to microglia and macrophages, these cells are highly responsive to IFN-γ, which upregulates the expression of enzymes, including IDO-1, producing neurotoxic KP metabolites such as quinolinic acid. These metabolites circulate systemically or are released locally in the brain and can contribute to the excitotoxic death of oligodendrocytes and neurons in neurological disease principally by virtue of their agonist activity at N-methyl-d-aspartic acid receptors. The latest evidence is presented and discussed. The enzymes that control the checkpoints in the KP represent an attractive therapeutic target, and consequently several KP inhibitors are currently in clinical trials for other neurological diseases, and hence may make suitable candidates for MS patients. Underpinning these drug discovery endeavors, in recent years, several advances have been made in how KP metabolites are assayed in various biological fluids, and tremendous advancements have been made in how specimens are imaged to determine disease progression and involvement of various cell types and molecules in MS.

Bcl11b: A New Piece to the Complex Puzzle of Amyotrophic Lateral Sclerosis Neuropathogenesis?

Amyotrophic lateral sclerosis (ALS) is an idiopathic, fatal, neurodegenerative disease of the human motor system. The pathogenesis of ALS is a topic of fascinating speculation and experimentation, with theories revolving around intracellular protein inclusions, mitochondrial structural issues, glutamate excitotoxicity and free radical formation. This review explores the rationale for the involvement of a novel protein, B-cell lymphoma/leukaemia 11b (Bcl11b) in ALS. Bcl11b is a multifunctional zinc finger protein transcription factor. It functions as both a transactivator and genetic suppressor, acting both directly, binding to promoter regions, and indirectly, binding to promoter-bound transcription factors. It has essential roles in the differentiation and growth of various cells in the central nervous system, immune system, integumentary system and cardiovascular system, to the extent that Bcl11b knockout mice are incompatible with extra-uterine life. It also has various roles in pathology including the suppression of latent retroviruses, thymic tumourigenesis and neurodegeneration. In particular its functions in neurodevelopment, viral latency and T-cell development suggest potential roles in ALS pathology.

P2X7 receptors mediate innate phagocytosis by human neural precursor cells and neuroblasts.

During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68(+) microglia and astrocytes mediate phagocytosis during target-dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple-marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live-cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum-free environment, doublecortin(+) (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7(high) DCX(low) hNPCs and P2X7(high) DCX(high) neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum-free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target-dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor.

Absence of platelet endothelial cell adhesion molecule 1, PECAM-1/CD31, in vivo increases resistance to Salmonella enterica serovar Typhimurium in mice.

PECAM-1/CD31 is known to regulate inflammatory responses and exhibit pro- and anti-inflammatory functions. This study was designed to determine the functional role of PECAM-1 in susceptibility to murine primary in vivo infection with Salmonella enterica serovar Typhimurium and in in vitro inflammatory responses of peritoneal macrophages. Lectin profiling showed that cellular PECAM-1 and recombinant human PECAM-1-Ig chimera contain high levels of mannose sugars and N-acetylglucosamine. Consistent with this carbohydrate pattern, both recombinant human and murine PECAM-1-Ig chimeras were shown to bind S. Typhimurium in a dose-dependent manner in vitro. Using oral and fecal-oral transmission models of S. Typhimurium SL1344 infection, PECAM-1(-/-) mice were found to be more resistant to S. Typhimurium infection than wild-type (WT) C57BL/6 mice. While fecal shedding of S. Typhimurium was comparable in wild-type and PECAM-1(-/-) mice, the PECAM-1-deficient mice had lower bacterial loads in systemic organs such as liver, spleen, and mesenteric lymph nodes than WT mice, suggesting that extraintestinal dissemination was reduced in the absence of PECAM-1. This reduced bacterial load correlated with reduced tumor necrosis factor (TNF), interleukin-6 (IL-6), and monocyte chemoattractant protein (MCP) levels in sera of PECAM-1(-/-) mice. Following in vitro stimulation of macrophages with either whole S. Typhimurium, lipopolysaccharide (LPS) (Toll-like receptor 4 [TLR4] ligand), or poly(I·C) (TLR3 ligand), production of TNF and IL-6 by PECAM-1(-/-) macrophages was reduced. Together, these results suggest that PECAM-1 may have multiple functions in resistance to infection with S. Typhimurium, including binding to host cells, extraintestinal spread to deeper tissues, and regulation of inflammatory cytokine production by infected macrophages.

A rapid cell counting method utilising acridine orange as a novel discriminating marker for both cultured astrocytes and microglia.

Cell culture analyses of growth, morphology and apoptosis commonly require counting of different cell types stained with antibodies to discriminate between them. Previously, we reported the use of l-Leucine methyl ester (l-LME) to prepare purified cultures of type 1 astrocytes with minimal microglia, and staining by GFAP and CD antibodies, respectively. Here, we demonstrate a novel use of acridine orange (AO) for rapid discrimination between these cell types using fluorescence microscopy. AO accumulates in the lysosomes and also binds strongly to nuclear DNA and cytoplasmic/nucleolar RNA. Microglia may contain abundant lysosomes due to known roles in homeostasis and immune response. AO staining of lysosomes was tested at a range of concentrations, and 2.5 microg/mL was most suitable. In agreement with previous reports, microglia treated with AO showed very intense yellow, orange or red granular cytoplasmic staining of lysosomes. Microglia contain a substantially higher number of lysosomes than astrocytes, which have a variable amount. We measured the microglia population at 5.14+/-0.50% in mixed cultures. Thus, these results show AO is a novel discriminatory marker, as microglia were easily observed and counted in clumps on top of the monolayer of astrocytes, providing a rapid alternative to time-consuming and costly antibody-based assays.