Astroglial Marker GFAP Research Articles

Immunophenotyping of peripheral blood mononuclear cells in tuberous sclerosis complex

Abstract Tuberous sclerosis complex (TSC) is a multisystem disorder caused by loss-of-function mutations in TSC1/2 proteins. This results in constitutive mTOR activation and predisposes to the development of benign tumours, with renal angiomyolipomas (AMLs) and tuber-related drug-resistant epilepsy (DRE) being major causes of morbidity and mortality in adults. Despite the central role of mTOR in leukocyte biology and the beneficial impact of mTOR inhibitors in TSC, the role of the immune system and inflammation in the development of tumours and DRE in people with TSC remains unknown. Using peripheral blood mononuclear cells (PBMCs) and serum from people with TSC, healthy controls (HC) and non-TSC DRE, we employed flow cytometry and multiplex assays to interrogate the immune profile associated with chronic loss of mTOR inhibition. We found that higher levels of the astroglial activation marker GFAP are elevated in TSC and show high intergenotype variability. We also observed differences in circulating immune cell populations, cytokines, chemokines and growth factors related to TSC. We found a higher proportion of B cells in parallel with increased total IgG but a reduction in IL-2 and IL-7 and a lower frequency of T cells. TSC with vs without AML showed differences in CD4:CD8 T cells ratio, monocytes and dendritic cells frequencies. Finally, loss of TSC1/2 affected T cell polarization, leading to an increased proportion of T c17CD8 +T cells, especially in subjects with DRE. Overall, our findings indicate TSC is associated with increased blood biomarkers for inflammation, glial activation, and distribution and profile of circulating PBMCs compared to controls.

Modulatory effects of vitamin B3 and its derivative on the levels of apoptotic and vascular regulators and cytoskeletal proteins in diabetic rat brain as signs of neuroprotection

BackgroundBeneficial effects of nicotinamide (NAm) and its derivates have been earlier shown in animal models of diabetes mellitus (DM), but the mechanisms of their neuroprotective activities are still largely unknown. The aim of the present study was to investigate if NAm and conjugate of nicotinic acid with gamma-aminobutyric acid (N-GABA) are able to modulate expression levels of apoptosis regulators, angiogenesis-related molecules, and specific cytoskeletal proteins in diabetic rat brain. MethodsAfter six weeks of streptozotocin induced type 1 DM, rats were daily administered either by NAm (100 mg/kg) or N-GABA (55 mg/kg) intraperitoneally for two weeks. Protein levels were assessed by western blot and immunohistochemistry. ResultsBoth NAm and N-GABA down-regulated NF-κB and Bax levels in diabetic rat brain, suggesting their anti-apoptotic activities. Tested compounds normalized VEGF and nNOS contents improving pro-angiogenic signaling reduced by hyperglycemia. Western blot showed marked up-regulation of astroglial marker GFAP and lowering neurofilament protein levels in DM group, confirmed immunohistochemically, indicating the development of reactive astrogliosis as a major response to diabetes-induced neurodegeneration. NAm had no effects on GFAP and Nf-L levels in the diabetic brain, while N-GABA increased their expression. Inversely, NAm and N-GABA dramatically reduced enhanced levels of GFAP and Nf-L found in the blood serum of diabetic rats, providing for the first time strong evidence for preserving blood-brain barrier integrity by studied compounds. ConclusionThus, NAm and N-GABA may exert neuroprotective effects by decreasing pro-apoptotic regulators levels and improving expression of angiogenic and cytoskeletal proteins impaired by hyperglycemia in rat brain.

Characterization and standardization of multiassay platforms for four commonly studied traumatic brain injury protein biomarkers: a TBI Endpoints Development Study.

Aim: There is a critical need to validate biofluid-based biomarkers as diagnostic and drug development tools for traumatic brain injury (TBI). As part of the TBI Endpoints Development Initiative, we identified four potentially predictive and pharmacodynamic biomarkers for TBI: astroglial markers GFAP and S100B and the neuronal markers UCH-L1 and Tau. Materials & methods: Several commonly used platforms for these four biomarkers were identified and compared on analytic performance and ability to detect gold standard recombinant protein antigens and to pool control versus TBI cerebrospinal fluid (CSF). Results: For each marker, only some assay formats could differentiate TBI CSF from the control CSF. Also, different assays for the same biomarker reported divergent biomarker values for the same biosamples. Conclusion: Due to the variability of TBI marker assay in performance and reported values, standardization strategies are recommended when comparing reported biomarker levels across assay platforms.

Circulatory Astrocyte and Neuronal EVs as Potential Biomarkers of Neurological Dysfunction in HIV-Infected Subjects and Alcohol/Tobacco Users.

The diagnosis of neurocognitive disorders associated with HIV infection, alcohol, and tobacco using CSF or neuroimaging are invasive or expensive methods, respectively. Therefore, extracellular vesicles (EVs) can serve as reliable noninvasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. Hence, our objective was to investigate the expression of astrocytic (GFAP) and neuronal (L1CAM) specific proteins in EVs circulated in the plasma of HIV subjects, with and without a history of alcohol consumption and tobacco smoking. The protein expression of GFAP (p < 0.01) was significantly enhanced in plasma EVs obtained from HIV-positive subjects and alcohol users compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. The L1CAM expression was found to be significantly elevated in cigarette smokers (p < 0.05). However, its expression was not found to be significant in HIV subjects and alcohol users. Both GFAP and L1CAM levels were not further elevated in HIV-positive alcohol or tobacco users compared to HIV-positive nonsubstance users. Taken together, our data demonstrate that the astrocytic and neuronal-specific markers (GFAP and L1CAM) can be packaged in EVs and circulate in plasma, which is further elevated in the presence of HIV infection, alcohol, and/or tobacco. Thus, the astroglial marker GFAP and neuronal marker L1CAM may represent potential biomarkers targeting neurological dysfunction upon HIV infection and/or alcohol/tobacco consumption.

The Effects of Neuropathic Pain on the State of Glial Cells and Hippocampal Neurogenesis in Old Animals

Abstract—We studied the effect of chronic neuropathic pain on the state of microglia and neurogenesis in the hippocampus in old mice. Neuropathic pain was induced by imposing three ligatures on the sciatic nerve of the right hind limb of animals. Testing behavior showed the presence of impaired working memory and locomotor activity in animals with neuropathic pain both in 1 week and 3 weeks after surgery. Behavioral disorders were accompanied by a decrease in hippocampal neurogenesis, as well as an increase in the expression of microglial markers Iba-1 and CD86 in the hippocampus of animals with a ligated sciatic nerve. In addition, the induction of neuropathic pain led to a change in the expression of astroglial markers S100β and GFAP in the hippocampus. The findings suggest that behavioral changes in neuropathic pain are accompanied by changes in the activity of microglia and astroglia, which leads to disruption of neurogenesis and a decrease in cognitive functions.

Abstract 5224: Conversion of human glioblastoma cells into neurons by neuronal transcription factors

Abstract Background: Glioblastoma is one of the most severe primary cancer types in the central nervous system. Because of genomic and epigenetic heterogeneity, GBM is infiltrative and resistant to conventional treatments such as radiation, chemotherapy and molecular targeting drugs. Glioblastoma often arises from astrocytes, which can be directly converted into neurons according to our earlier work, so we hypothesize that glioblastoma cells might also be converted into non-proliferating neurons. This trans-differentiation therapy might provide a unique approach for glioblastoma treatment. Methods: NeuroD1 was chosen as one of the candidate factors in this study because we have shown its critical roles in astrocyte-to-neuron conversion. Neurog2 and Ascl1 were also tested to understand possible different conversion mechanisms. Single transcription factor or GFP was overexpressed via retrovirus in human glioblastoma cells. Twelve hours after virus infection, culture medium was changed into differentiation medium containing neurotropic factors for neuronal maturation. Immuostainng and other tests were conducted at different days post infection. Results: Retrovirus yielded high infection efficiency in fast-proliferating glioblastoma cells. All three factors tested were capable of converting glioblastoma cells into neuron-like cells efficiently. Besides morphological change, robust pan-neuronal markers were expressed during the conversion, such as immature neuronal markers DCX, Tuj1 and mature neuronal makers MAP2, NeuN. Majority of the converted cells from glioma were immunopositive for glutamatergic neuron marker vGluT1 and hippocampal neuron marker Prox1. Reactive astroglial marker GFAP decreased after conversion, but cancer marker EGFR and IL13Ra2 remained during conversion. Cell proliferation was inhibited during conversion indicated by Ki67 and BrdU. Robust synaptic puncta along dendrites were found in glioma-converted cells, indicated by SV2 immunostaining. Patch-clamp recordings revealed that most of the converted neurons could fire multiple action potentials or single action potential. Conclusion: Our data suggest that several neuronal transcription factors are capable to convert human glioblastoma cells into neuron-like cells efficiently. The converted cells obtained a variety of neuron-specific markers with functional synaptic networks and active electrophysiological properties. This neuronal conversion was also confirmed by reduction of reactive astroglial marker GFAP. Although some cancer markers remain in the converted neurons, glioblastoma cells stopped proliferating once being converted. In summary, our study suggests that converting human glioblastoma cells into neurons could be a potential therapeutic approach for glioblastoma treatment to at least control cancer cell proliferation and inhibit tumor progression. Citation Format: Xin Wang, Zifei Pei, Aasma Hossain, Tania T. Barnatan, Yuting Bai, Gong Chen. Conversion of human glioblastoma cells into neurons by neuronal transcription factors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5224.

Astrogliogenesis in human fetal brain: complex spatiotemporal immunoreactivity patterns of GFAP, S100, AQP4 and YKL-40.

The astroglial lineage consists of heterogeneous cells instrumental for normal brain development, function and repair. Unfortunately, this heterogeneity complicates research in the field, which suffers from lack of truly specific and sensitive astroglial markers. Nevertheless, single astroglial markers are often used to describe astrocytes in different settings. We therefore investigated and compared spatiotemporal patterns of immunoreactivity in developing human brain from 12 to 21 weeks post conception and publicly available RNA expression data for four established and potential astroglial markers - GFAP, S100, AQP4 and YKL-40. In the hippocampal region, we also screened for C3, a complement component highly expressed in A1-reactive astrocytes. We found diverging partly overlapping patterns of the established astroglial markers GFAP, S100 and AQP4, confirming that none of these markers can fully describe and discriminate different developmental forms and subpopulations of astrocytes in human developing brain, although AQP4 seems to be the most sensitive and specific marker for the astroglial lineage at midgestation. AQP4 characterizes a brain-wide water transport system in cerebral cortex with regional differences in immunoreactivity at midgestation. AQP4 distinguishes a vast proportion of astrocytes and subpopulations of radial glial cells destined for the astroglial lineage, including astrocytes determined for the future glia limitans and apical truncated radial glial cells in ganglionic eminences, devoid of GFAP and S100. YKL-40 and C3d, previously found in reactive astrocytes, stain different subpopulations of astrocytes/astroglial progenitors in developing hippocampus at midgestation and may characterize specific subpopulations of 'developmental astrocytes'. Our results clearly reflect that lack of pan-astrocytic markers necessitates the consideration of time, region, context and aim when choosing appropriate astroglial markers.

Neuroinflammation in the dorsolateral prefrontal cortex in elderly chronic schizophrenia

Cognitive deterioration and symptom progression occur in schizophrenia over the course of the disorder. A dysfunction of the immune system/neuroinflammatory pathways has been linked to schizophrenia (SZ). These altered processes in the dorsolateral prefrontal cortex (DLPFC) could contribute to the worsening of the deficits. However, limited studies are available in this brain region in elderly population with long-term treatments. In this study, we explore the possible deregulation of 21 key genes involved in immune homeostasis, including pro- and anti-inflammatory cytokines, cytokine modulators (toll-like receptors, colony-stimulating factors, and members of the complement system) and microglial and astroglial markers in the DLPFC in elderly chronic schizophrenia. We used quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) on extracts from postmortem DLPFC of elderly subjects with chronic SZ (n = 14) compared to healthy control individuals (n = 14). We report that CSF1R, TLR4, IL6, TNFα, TNFRSF1A, IL10, IL10RA, IL10RB, and CD68 were down-regulated in elderly SZ subjects. Moreover, we found that the expression levels of all the altered inflammatory genes in SZ correlated with the microglial marker CD68. However, no associations were found with the astroglial marker GFAP. This study reveals a decrease in the gene expression of cytokines and immune response/inflammation mediators in the DLPFC of elderly subjects with chronic schizophrenia, supporting the idea of a dysfunction of these processes in aged patients and its possible relationship with active microglia abundance. These findings include elements that might contribute to the cognitive decline and symptom progression linked to DLPFC functioning at advanced stages of the disease.

YKL-40 (Chitinase 3-like I) is expressed in a subset of astrocytes in Alzheimer\u2019s disease and other tauopathies

BackgroundThe innate immune system is known to be involved early in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative disorders. The inflammatory response in the central nervous system can be measured postmortem or through a series of inflammatory mediator surrogates. YKL-40 (also named Chitinase-3-like I) has been frequently investigated in body fluids as a surrogate marker of neuroinflammation in AD and other neurological disorders. However, the expression pattern of YKL-40 in the human brain with neurodegenerative pathology remains poorly investigated. Our aim was to study the cellular expression pattern of YKL-40 in the brain of patients with clinical and neuropathological criteria for AD (n = 11); three non-AD tauopathies: Pick’s disease (PiD; n = 8), corticobasal degeneration (CBD; n = 8) and progressive supranuclear palsy (PSP; n = 9) and a group of neurologically healthy controls (n = 6).MethodsSemiquantitative neuropathological evaluation and quantitative confocal triple immunofluorescence studies were performed. An in-house algorithm was used to detect and quantify pathology burden of random regions of interest on a full tissue-section scan. Kruskal-Wallis and Dunn’s multiple comparison tests were performed for colocalization and quantification analyses.ResultsWe found that brain YKL-40 immunoreactivity was observed in a subset of astrocytes in all four diseases and in controls. There was a strong colocalization between YKL-40 and the astroglial marker GFAP but not with neuronal nor microglial markers. Intriguingly, YKL-40-positive astrocytes were tau-negative in PSP, CBD and PiD. The number of YKL-40-positive astrocytes was increased in tauopathies compared with that in controls. A positive correlation was found between YKL-40 and tau immunoreactivities.ConclusionsThis study confirms that YKL-40 is expressed by a subset of astrocytes in AD and other tauopathies. YKL-40 expression is elevated in several neurodegenerative conditions and correlates with tau pathology.

Sex-Dependent Glial Signaling in Pathological Pain: Distinct Roles of Spinal Microglia and Astrocytes.

Increasing evidence suggests that spinal microglia regulate pathological pain in males. In this study, we investigated the effects of several microglial and astroglial modulators on inflammatory and neuropathic pain following intrathecal injection in male and female mice. These modulators were the microglial inhibitors minocycline and ZVEID (a caspase-6 inhibitor) and the astroglial inhibitors L-α-aminoadipate (L-AA, an astroglial toxin) and carbenoxolone (a connexin 43 inhibitor), as well as U0126 (an ERK kinase inhibitor) and D-JNKI-1 (a c-Jun N-terminal kinase inhibitor). We found that spinal administration of minocycline or ZVEID, or Caspase6 deletion, reduced formalin-induced inflammatory and nerve injury-induced neuropathic pain primarily in male mice. In contrast, intrathecal L-AA reduced neuropathic pain but not inflammatory pain in both sexes. Intrathecal U0126 and D-JNKI-1 reduced neuropathic pain in both sexes. Nerve injury caused spinal upregulation of the astroglial markers GFAP and Connexin 43 in both sexes. Collectively, our data confirmed male-dominant microglial signaling but also revealed sex-independent astroglial signaling in the spinal cord in inflammatory and neuropathic pain.

Protein Characterization of Extracellular Microvesicles/Exosomes Released from Cytotoxin-Challenged Rat Cerebrocortical Mixed Culture and Mouse N2a Cells.

A number of neuronal and glial proteins were previously found to be released in free-standing soluble form from cultured brain cells into cell-conditioned media. Here, we sought to examine if similar proteins are also contained in neural and astroglial cell-released extracellular microvesicles/exosomes (MV/E). In this study, MV/E were isolated from cell-conditioned media from control and cytotoxin-challenged rat cerebrocortical mixed culture (CTX) and mouse neuroblastoma N2a cells. Cytotoxin challenges included pro-necrosis calcium ionophore A23187, pro-apoptosis staurosporine (STS), and excitotoxin N-methyl-D-aspartate. Based on established nanoparticle characterization method (dynamic light scattering, NanoTracker, and transmission electron microscopy), we confirmed that these released vesicles are in fact characteristic representation of MV/E by morphology (lipid bilayered vesicles) and by particle size (132-142nm for CTX and 49-77nm for N2a cells). We indeed identified neural cell body protein UCH-L1, axonal injury marker αII-spectrin and its breakdown products (SBDPs), astroglial markers GFAP and its breakdown products (GFAP-BDP), dendritic protein BIII-tubulin, synaptic protein synaptophysin, and exosome marker Alix in microvesicles from CTX and/or N2a cells. Furthermore, SBDPs, GFAP-BDP, UCH-L1, and synaptophysin are especially dominant in MV/E isolated from cytotoxin-treated CTX cells. Similarly, SBDPs, βIII-tubulin, and UCH-L1 are more prominently observed in cytotoxin-challenged N2a cells. Lastly, when isolated MV/E from A23187- or STS-challenged N2a cells were introduced to healthy N2a culture, they are capable of evoking cytotoxicity in the latter. Taken together, our study identified that microvesicles/exosomes isolated form healthy and injured brain cells contain certain neural and astroglial proteins, as well as possibly other cytotoxic factors that are capable of propagating cytotoxic effects.

Intranasal Oncolytic Virotherapy with CXCR4-Enhanced Stem Cells Extends Survival in Mouse Model of Glioma.

SummaryThe challenges to effective drug delivery to brain tumors are twofold: (1) there is a lack of non-invasive methods of local delivery and (2) the blood-brain barrier limits systemic delivery. Intranasal delivery of therapeutics to the brain overcomes both challenges. In mouse model of malignant glioma, we observed that a small fraction of intranasally delivered neural stem cells (NSCs) can migrate to the brain tumor site. Here, we demonstrate that hypoxic preconditioning or overexpression of CXCR4 significantly enhances the tumor-targeting ability of NSCs, but without altering their phenotype only in genetically modified NSCs. Modified NSCs deliver oncolytic virus to glioma more efficiently and extend survival of experimental animals in the context of radiotherapy. Our findings indicate that intranasal delivery of stem cell-based therapeutics could be optimized for future clinical applications, and allow for safe and repeated administration of biological therapies to brain tumors and other CNS disorders.

Inhibition of glial proliferation, promotion of axonal growth and myelin production by synthetic glycolipid: A new approach for spinal cord injury treatment.

After spinal cord injury (SCI) a glial scar is generated in the area affected that forms a barrier for axon growth and myelination, preventing functional recovery. Recently, we have described a synthetic glycolipid (IG20) that inhibited proliferation of human glioma cells. We show now that IG20 inhibited the proliferation of astrocytes and microglial cells, the principal cellular components of the glial scar, and promoting axonal outgrowth and myelin production in vitro. Glial cells were inhibited with IG20 (IC50≈10 μM) and studied by RT-PCR, Western Blotting, immunoprecipitation and fluorescence microscopy. Axonal outgrowth in dorsal root ganglia (DRG) and myelin production by oligodendrocytes were analyzed by immunocytochemistry. Adult rats were assayed in spinal cord contusion model and the recovery of treated animals (n = 6) and controls (n = 6) was followed. The IG20 was localized in the cytosol of glial cells, forming a complex with RhoGDIα, a regulator of RhoGTPases. Treatment of astroglial cultures with IG20 increase the expression of BDNF receptor genes (TrkBT1, TrkB Full). IG20 reduced the astroglial marker GFAP, while increasing production of myelin basic protein in oligodendrocytes and promoted axonal outgrowth from DRG neurons. Local injection of IG20, near a spinal cord contusion, promoted the recovery of lesioned animals analyzed by BBB test (P < 0.05). We propose that inhibition of astrocytes and microglia by IG20 could be diminished the glial scar formation, inducing the re-growth and myelination of axons, these elements constitute a new approach for SCI therapy.

Prominin-1 (CD133) defines both stem and non-stem cell populations in CNS development and gliomas.

Prominin-1 (CD133) is a commonly used cancer stem cell marker in central nervous system (CNS) tumors including glioblastoma (GBM). Expression of Prom1 in cancer is thought to parallel expression and function in normal stem cells. Using RNA in situ hybridization and antibody tools capable of detecting multiple isoforms of Prom1, we find evidence for two distinct Prom1 cell populations in mouse brain. Prom1 RNA is first expressed in stem/progenitor cells of the ventricular zone in embryonic brain. Conversely, in adult mouse brain Prom1 RNA is low in SVZ/SGZ stem cell zones but high in a rare but widely distributed cell population (Prom1hi). Lineage marker analysis reveals Prom1hi cells are Olig2+Sox2+ glia but Olig1/2 knockout mice lacking oligodendroglia retain Prom1hi cells. Bromodeoxyuridine labeling identifies Prom1hi as slow-dividing distributed progenitors distinct from NG2+Olig2+ oligodendrocyte progenitors. In adult human brain, PROM1 cells are rarely positive for OLIG2, but express astroglial markers GFAP and SOX2. Variability of PROM1 expression levels in human GBM and patient-derived xenografts (PDX) – from no expression to strong, uniform expression – highlights that PROM1 may not always be associated with or restricted to cancer stem cells. TCGA and PDX data show that high expression of PROM1 correlates with poor overall survival. Within proneural subclass tumors, high PROM1 expression correlates inversely with IDH1 (R132H) mutation. These findings support PROM1 as a tumor cell-intrinsic marker related to GBM survival, independent of its stem cell properties, and highlight potentially divergent roles for this protein in normal mouse and human glia.

Early postnatal GFAP-expressing cells produce multilineage progeny in cerebrum and astrocytes in cerebellum of adult mice

Early postnatal GFAP-expressing cells are thought to be immature astrocytes. However, it is not clear if they possess multilineage capacity and if they can generate different lineages (astrocytes, neurons and oligodendrocytes) in the brain of adult mice.In order to identify the fate of astroglial cells in the postnatal brain, hGFAP-Cre-ERT2 transgenic mice were crossed with the R26R Cre reporter mouse strains which exhibit constitutive expression of β-galactosidase (β-gal). Mice carrying the hGFAP-Cre-ERT2/R26R transgene were treated with Tamoxifen to induce Cre recombination in astroglial cells at postnatal (P) day 6 and Cre recombinase-expressing cells were identified by X-gal staining. Immunohistochemical staining was used to identify the type(s) of these reporter-tagged cells. Sixty days after recombination, X-gal-positive cells in different cerebral regions of the adult mice expressed the astroglial markers Blbp and GFAP, the neuronal marker NeuN, the oligodendrocyte precursor cell marker NG2 and the mature oligodendrocyte marker CC1. X-gal-positive cells in the cerebellum coexpressed the astroglial marker Blbp, but not the granule cell marker NeuN, Purkinje cell marker Calbindin or oligodendrocyte precursor cell marker NG2.Our genetic fate mapping data demonstrated that early postnatal GFAP-positive cells possessed multilineage potential and eventually differentiated into neurons, astrocytes, and oligodendrocyte precursor cells in the cerebrum and into astrocytes (including Bergmann glia) in the cerebellum of adult mice.

Diversity of Neural Precursor Cell Types in the Prenatal Macaque Cerebral Cortex Exists Largely within the Astroglial Cell Lineage

The germinal zones of the embryonic macaque neocortex comprise the ventricular zone (VZ) and the subventricular zone (SVZ). The mammalian SVZ is subdivided into an inner SVZ and an outer SVZ, with the outer SVZ being particularly large in primates. The existence of distinct precursor cell types in the neocortical proliferative zones was inferred over 100 years ago and recent evidence supports this concept. Precursor cells exhibiting diverse morphologies, patterns of transcription factor expression, and fate potential have been identified in the neocortical proliferative zones. Neurogenic precursor cells are thought to exhibit characteristics of glial cells, but the existence of neurogenic precursor cells that do not share glial specific properties has also been proposed. Therefore, one question that remains is whether neural precursor cells in the prenatal neocortex belong within the astroglial cell class, as they do in neurogenic regions of the adult neocortex, or instead include a diverse collection of precursor cells belonging to distinct cell classes. We examined the expression of astroglial markers by mitotic precursor cells in the telencephalon of prenatal macaque and human. We show that in the dorsal neocortex all mitotic cells at the surface of the ventricle, and all Pax6+ and Tbr2+ mitotic cells in the proliferative zones, express the astroglial marker GFAP. The majority of mitotic cells undergoing division away from the ventricle express GFAP, and many of the GFAP-negative mitoses express markers of cells derived from the ventral telencephalon or extracortical sites. In contrast, a markedly lower proportion of precursor cells express GFAP in the ganglionic eminence. In conclusion, we propose that the heterogeneity of neural precursor cells in the dorsal cerebral cortex develops within the GFAP+ astroglial cell class.

Changes in the expression of astroglial and microglial markers in the hippocampus of rats adapted to chronic stress and the effects of panthenol

We studied the morphological changes and expression of the astroglial and microglial markers in the sensorimotor cortex and hippocampus of rats that were subjected to chronic stress. Chronic neurotization was associated with the appearance of a large number of damaged neurons in layer V of the sensorimotor cortex and the pyramidal layer of the hippocampus. These changes were considerably expressed in the CA3 field. A neurosis-like state in rats was accompanied by an increased expression of microglial markers in the hippocampus and this effect was evident even 1 month after the end of stress. The expression of the astroglial marker GFAP decreased in the CA3 hippocampal field. Treatment of animals with the pantothenic acid derivative panthenol stabilized the glial response to chronic stress; however, its effects were not long-lasting and did not prevent activation of microglia during the period after stress.

Neuroprotective Effects of N-Acetyl-Cysteine and Acetyl-L-Carnitine after Spinal Cord Injury in Adult Rats

Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4–L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.

Abstract 4319: Effects of chemotherapeutics on organotypic corticostriatal slice cultures identified by a new panel of fluorescent and immunohistochemical markers

Abstract Effects of chemotherapeutics on glioma cell lines and spheroids are usually investigated without evaluating the effects of even very high concentrations of chemotherapeutics on normal brain tissue. To perform such investigations, the aim of the present study was to establish a panel of markers for detection of general cell death and more specific neuronal and glial degeneration induced by chemotherapeutics in organotypic rat corticostriatal slice cultures. Organotypic rat corticostriatal slice cultures were exposed to the alkylating agents temozolomide and nimustine, the tyrosine kinase inhibitor imatinib mesylate and the microtubule destabilizing agent vincristine. Densitometric measurements of uptake of the fluorescent dye propidium iodide were used for quantifying cellular degeneration. Moreover, paraffin sections were HE stained and immunohistochemically stained for the neuronal marker MAP2, the astroglial marker GFAP, and the oligodendrocyte marker p25α. The results showed that clinical drug concentrations were non-toxic. However, a time dependent increase in PI uptake was observed for supratherapeutic concentrations of the drugs, except for temozolomide, where no toxicity was observed at all. Corresponding immunostaining showed loss of MAP2 staining and increased expression of GFAP and p25α for cultures exposed to 1000 nM vincristine. Cultures exposed to supratherapeutic concentrations of nimustine and imatinib mesylate disintegrated, leaving no tissue for histology. In conclusion, corticostriatal slice cultures and the established panel of markers represent an excellent tool for detecting toxicity by chemotherapeutics. Toxicity was not detected at clinical concentrations, but high concentrations with toxic effects were identified suggesting that some of the earlier identified anti-cancer effects in the literature are general cytotoxic effects and not specific anti-cancer effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4319. doi:10.1158/1538-7445.AM2011-4319

Expression of purinergic P2X7 receptor in rat brain during the symptomatic phase of experimental autoimmune encephalomyelitis and after recovery of neurological deficits.

Purinergic ionotropic P2X(7) receptor is widely distributed in brain. Strong evidence suggests that this receptor is related to inflammatory and neurodegenerative changes in many pathological states of central nervous system (CNS), including multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) is the commonly used animal model of MS. In this study we investigate the expression of P2X(7)R protein in rat brain in the symptomatic phase of EAE (day 10 post immunization) and after reversion of neurological symptoms (day 20 p.i.). We found the increased level of P2X(7)R protein in brain homogenates of EAE rats in both examined time windows. Immunohistochemical study revealed enhanced receptor's immunoreactivity. Immunoblots done with isolated cellular brain fractions indicated that the P2X(7)R overexpression is related to synaptosomal fraction in the symptomatic phase and to the glial (GPV) fraction in the recovery phase of EAE. Concomitantly, we noticed overexpression of astroglial marker GFAP in brain homogenates and astroglial fraction (GPV), so as its enhanced immunoreactivity in brain sections (10 days p.i.) which did not decline to control values in the recovery phase, similarly to P2X(7)R expression. Results suggest the involvement of P2X(7)R-mediated signaling in the pathomechanisms of EAE with the possible relevance of astrocytic pool of cells.