Single-cell ELISA and flow cytometry as methods for highlighting potential neuronal and astrocytic toxicant specificity

The timeline imposed by recent worldwide chemical legislation is not amenable to conventional in vivo toxicity testing, requiring the development of rapid, economical in vitro screening strategies which have acceptable predictive capacities. When acquiring regulatory neurotoxicity data, distinction on whether a toxic agent affects neurons and/or astrocytes is essential. This study evaluated neurofilament (NF) and glial fibrillary acidic protein (GFAP) directed single-cell (S-C) ELISA and flow cytometry as methods for distinguishing cell-specific cytoskeletal responses, using the established human NT2 neuronal/astrocytic (NT2.N/A) co-culture model and a range of neurotoxic (acrylamide, atropine, caffeine, chloroquine, nicotine) and non-neurotoxic (chloramphenicol, rifampicin, verapamil) test chemicals. NF and GFAP directed flow cytometry was able to identify several of the test chemicals as being specifically neurotoxic (chloroquine, nicotine) or astrocytoxic (atropine, chloramphenicol) via quantification of cell death in the NT2.N/A model at cytotoxic concentrations using the resazurin cytotoxicity assay. Those neurotoxicants with low associated cytotoxicity are the most significant in terms of potential hazard to the human nervous system. The NF and GFAP directed S-C ELISA data predominantly demonstrated the known neurotoxicants only to affect the neuronal and/or astrocytic cytoskeleton in the NT2.N/A cell model at concentrations below those affecting cell viability. This report concluded that NF and GFAP directed S-C ELISA and flow cytometric methods may prove to be valuable additions to an in vitro screening strategy for differentiating cytotoxicity from specific neuronal and/or astrocytic toxicity. Further work using the NT2.N/A model and a broader array of toxicants is appropriate in order to confirm the applicability of these methods.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

The retrograde delivery of adenovirus vector carrying the gene for brain-derived neurotrophic factor protects neurons and oligodendrocytes from apoptosis in the chronically compressed spinal cord of twy/twy mice

STUDY DESIGN: The twy/twy mouse undergoes spontaneous chronic mechanical compression of the spinal cord; this in vivo model system was used to examine the effects of retrograde adenovirus (adenoviral vector [AdV])-mediated brain-derived neurotrophic factor (BDNF) gene delivery to spinal neural cells. OBJECTIVE: To investigate the targeting and potential neuroprotective effect of retrograde AdV-mediated BDNF gene transfection in the chronically compressed spinal cord in terms of prevention of apoptosis of neurons and oligodendrocytes. SUMMARY OF BACKGROUND DATA: Several studies have investigated the neuroprotective effects of neurotrophins, including BDNF, in spinal cord injury. However, no report has described the effects of retrograde neurotrophic factor gene delivery in compressed spinal cords, including gene targeting and the potential to prevent neural cell apoptosis. METHODS: AdV-BDNF or AdV-LacZ (as a control gene) was injected into the bilateral sternomastoid muscles of 18-week old twy/twy mice for retrograde gene delivery via the spinal accessory motor neurons. Heterozygous Institute of Cancer Research mice (+/twy), which do not undergo spontaneous spinal compression, were used as a control for the effects of such compression on gene delivery. The localization and cell specificity of ß-galactosidase expression (produced by LacZ gene transfection) and BDNF expression in the spinal cord were examined by coimmunofluorescence staining for neural cell markers (NeuN, neurons; reactive immunology protein, oligodendrocytes; glial fibrillary acidic protein, astrocytes; OX-42, microglia) 4 weeks after gene injection. The possible neuroprotection afforded by retrograde AdV-BDNF gene delivery versus AdV-LacZ-transfected control mice was assessed by scoring the prevalence of apoptotic cells (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells) and immunoreactivity to active caspases -3, -8, and -9, p75, neurofilament 200 kD (NF), and for the oligodendroglial progenitor marker, NG2. RESULTS.: Four weeks after injection, the retrograde delivery of the LacZ marker gene was identified in cervical spinal neurons and some glial cells, including oligodendrocytes in the white matter of the spinal cord, in both the twy/twy mouse and the heterozygous Institute of Cancer Research mouse (+/twy). In the compressed spinal cord of twy/twy mouse, AdV-BDNF gene transfection resulted in a significant decrease in the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cells present in the spinal cord and a downregulation in the caspase apoptotic pathway compared with AdV-LacZ (control) gene transfection. There was a marked and significant increase in the areas of the spinal cord of AdV-BDNF-injected mice that were NF- and NG2-immunopositive compared with AdV-LacZ-injected mice, indicating the increased presence of neurons and oligodendrocytes in response to BDNF transfection. CONCLUSION: Our results demonstrate that targeted retrograde BDNF gene delivery suppresses apoptosis in neurons and oligodendrocytes in the chronically compressed spinal cord of twy/twy mouse. Further work is required to establish whether this method of gene delivery may provide neuroprotective effects in other situations of compressive spinal cord injury.

Assessment of the astrogliotic responses of three human astrocytoma cell lines to ethanol, trimethyltin chloride and acrylamide

The astrogliotic responses of the CCF-STTG1, U251-MG, and U373-MG human astrocytoma lines were determined after exposure to ethanol, trimethyltin chloride (TMTC), and acrylamide over 4, 16, and 24 h. Basal glial fibrillary acidic protein (GFAP) expression in the U-251MG and U373-MG cells was 10-fold greater than the CCF-STGG1 line. Ethanol treatment over 24 h, but not at 4 and 16 h, resulted in significant increases in GFAP in all three glioma lines at sub-cytotoxic levels; the GFAP responses in the CCF-STTG1 line were the most sensitive, as concentrations of 0.1 and 1 mM led to increases in GFAP expression compared with control of 56.8 ± 15.7 and 58.9 ± 11.5%, respectively (P < 0.05). Treatment with TMTC (1 μM) over 4 h showed elevated GFAP expression in the U251-MG cell line to 28.0 ± 15.7% above control levels (P < 0.01), but not in the other U373-MG or CCF-STTG1 cells. At 4 h, MTT turnover was markedly increased compared with control, particularly in the U373-MG line at concentrations as low as 1 μM (17.1 ± 2.3%; P < 0.01). TMTC exposure over 16 and 24 h resulted in reduction in GFAP expression in all three lines at concentrations; at 24 h incubation, the reduction was >50% (P < 0.01). There were no changes in GFAP expression or MTT turnover in response to acrylamide except at the highest concentration ranges of 10-100 mM. This study underlines the significance of period of exposure, as well as toxin concentration in astrocytoma cellular response to toxic pressure. © 2007 Elsevier Ireland Ltd. All rights reserved.