Morphometric assessment of toxicant induced neuronal degeneration in full and restricted contact co-cultures of embryonic cortical rat neurons and astrocytes: Using m-Dinitrobezene as a model neurotoxicant

Abstract

With m-Dinitrobenzene (m-DNB) as a selected model neurotoxicant, we demonstrate how to assess neurotoxicity, using morphology based measurement of neurite degeneration, in a conventional “full-contact” and a modern “restricted-contact” co-culture of rat cortical neurons and astrocytes. In the “full-contact” co-culture, neurons and astrocytes in complete physical contact are “globally” exposed to m-DNB. A newly emergent “restricted-contact” co-culture is attained with a microfluidic device that polarizes neuron somas and neurites into separate compartments, and the neurite compartment is “selectively” exposed to m-DNB. Morphometric analysis of the neuronal area revealed that m-DNB exposure produced no significant change in mean neuronal cell area in “full-contact” co-cultures, whereas a significant decrease was observed for neuron monocultures. Neurite elaboration into a neurite exclusive compartment in a compartmentalized microfluidic device, for both monocultures (no astrocytes) and “restricted” co-cultures (astrocytes touching neurites), decreased with exposure to increasing concentrations of m-DNB, but the average neurite area was higher in co-cultures. By using co-culture systems that more closely approach biological and architectural complexities, and the directionality of exposure found in the brain, this study provides a methodological foundation for unraveling the role of physical contact between astrocytes and neurons in mitigating the toxic effects of chemicals such as m-DNB.

Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds

“…The findings of the present study provide further support for other published literature 6,7,[23][24][25][26][27] demonstrating the protective/ameliorative effects to toxins/toxicants of coculturing neuronal-astrocyte cells, and specifically does so in a transwell/restricted contact model (as applied in this study), for which there are limited other publications, 6,25,28 as well doing so using human-derived cells 8,9,29 instead of rodent-derived cells.…”

“…These findings are in agreement with literature data, although still limited in this area, on neuroprotection effects by astrocytes on neurons, that evaluated different end points and compounds (eg, 6-hydroxydopamine, tributyltin, trimethyltin, 2,5-hexanedione, MeHg, MGO, m-dinitrobenzene, and small iron oxide particles) using different co-culture models. 6,7,[23][24][25][26][27] In particular, these CNS co-culture models apply primary rodent astrocytes and neurons or mixture of primary/ immortalized rodent astrocytes and human neurons mainly by using 2 methods of co-culture:…”

Thermoplasmonic neural chip platform for in situ manipulation of neuronal connections in vitro

“…At power densities of 68 and 101 W/mm 2 , however, morphological neurite alterations occurred in and around the NIR-focused region. The observed morphological changes were neurite transection, beading, and fragmentation, which were used as indicators of neurite injury and degeneration in cultured nerve cells [25][26][27] . At the highest power level of 101 W/mm 2 , it was found that the surrounding agarose hydrogel also melted due to the thermoplasmonic heat.…”

Morphometric and computational assessments to evaluate neuron survival and maturation within compartmentalized microfluidic devices: The influence of design variation on diffusion-driven nutrient transport

“…Microfluidic devices with semblance to Teflon Campenot chambers, parse soma and neurites into separate compartments linked by a series of grooves that serve as a passageway for neurites. These unique segregated culture schemes have been broadly adapted as in vitro models for axonal injury [1], axonal transport [2], and neurite-glia interactions [3–5]. However, slight deviations in device geometry among these Campenot-like microfluidic devices induce vastly different nutrient transport profiles that influence cell viability and neurite extension, and there is limited work offering a detailed rationale for these observations.…”