Single-Cell ELISA and Flow Cytometry as Methods for Highlighting Potential Neuronal and Astrocytic Toxicant Specificity

Abstract

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.

NT2 Derived Neuronal and Astrocytic Network Signalling

“…The proportion of cell types produced by this method in this study were in agreement with previously published values (33±4% neurons and 63±4% astrocytes) [23]. NT2A cultures in our hands typically survive >9 months (unpublished data).…”

“…It has also been found that NT2A cells are coupled via connexin 43 containing gap junctions [20]. In addition, in our laboratory we have used the NT2N/NT2A co-culture system, to demonstrate that astrocytes confer significantly increased neuronal resilience to chemical toxicity and that neurons and astrocytes display markedly differential sensitivities to a number of CNS toxins [22], [23]. Such findings therefore provide evidence of the distinct functionality of NT2A cells.…”

The effects of the fungicides fenhexamid and myclobutanil on SH-SY5Y and U-251 MG human cell lines

“…Taken together, our observations of the pesticides' impact on the expression on cell cycle regulating genes, along with the chronic nature of "real world" low level exposure, suggests that the potential risks of these agents to man remain to be quantified and thus warrant further investigation, ideally using a post mitotic model of neurons and astrocytes amenable to chronic investigation (Woehrling et al, 2011;Hill et al, 2013).…”

A critical review of cell culture strategies for modelling intracortical brain implant material reactions

“…7A and B) [137], B35s [138] and SH-SY5Y [139] cells. Neural cell lines, such as the human NT2 cell line (hNT) [140], are often derived from pluripotent teratocarcinoma cells and can be influenced to differentiate into CNS neurons. Primary neural cell cultures are derived from fetal CNS tissues or from embryonic chick cortex ( Fig.…”