Establishment of a Simple and Versatile Evaporation Compensation Model for in vitro Chronic Ethanol Treatment: Impact on Neuronal Viability

Abstract

Alcohol overconsumption is a major cause of preventable mental disorders and death in the United States and around the world. The pathogenesis of alcohol dependence, abuse, and toxicity to the central nervous system remains incompletely understood. In vitro and cell culture-based models have been highly valuable in studying the molecular and cellular mechanisms underlying the contribution of individual CNS cell types to ethanol's effects on the brain. However, conventional cell culture model systems carry the inherent disadvantage of rapid loss of ethanol due to evaporation following a bolus addition at the start of the treatment. We have established a multi-well cell culture plate-based ethanol evaporation compensation model that utilizes the inter-well space as a reservoir to compensate for the evaporative loss of ethanol in the cell treatment wells. Following a single bolus addition at the start of the treatment, ethanol concentration rapidly decreased over time. Through compensation using the multi-well plate platform, maintenance of ethanol concentrations ranging from 10-100 mM was achieved for up to 72 hours in a cell-free system. Greater effects in ethanol-induced decrease in neuronal cell viability were observed with than without compensation. Our method effectively compensates for the evaporative loss of ethanol typically observed in the traditional method. This method provides an economic, simple and effective in vitro model system for ethanol treatment over an extended timeframe where maintenance of a relatively constant concentration of ethanol is desired.

Keywords: cell culture model; chronic; compensation; ethanol; evaporation; neurons; viability.

Figure 1.. Time-dependent evaporation of ethanol with a starting calculated concentration of 10 mM and effects of compensation.

A. At the start of the experiment (0 hour), ethanol (10 mM) was added to wells of 6-well culture plates (3 ml of culture media/well) and the plates were incubated in a CO₂ cell culture incubator in a humidified environment with 5% CO₂ and 95% air. B. At the indicated timed points, ethanol concentrations in the corner and middle wells were measured (black lines). In separate 6-well plates, additional ethanol was added to the inter-well space to 40 mM for evaporation compensation (4X Comp) at the start (blue lines, +) or at the start and again at the 48-hour time point (4X Comp, red symbols, ++) and ethanol concentrations were measured at the indicated timed points. The green dash line indicates the target ethanol concentration (10 mM). Results are expressed as mean ± SEM of the average of the two middle and four corner wells from 3 independent experiments. * and **, p < 0.05 and 0.005 respectively compared to the calculated 10 mM starting ethanol concentration.

Figure 2.. Ethanol evaporation and effects of compensation with a starting ethanol concentration of 25 mM.

The magnitude of evaporation (black lines) was determined at the indicated timed points following a single bolus addition of a calculated concentration of 25 mM ethanol to the culture wells. The effects of compensation on the ethanol concentrations in the culture wells were evaluated by the addition of 125 mM ethanol at the start (5X Comp, blue lines, +) or 100 mM ethanol again at the 48-hour time point (4X Comp, red lines/symbols, ++) were evaluated. Results are expressed as mean ± SEM from 3 independent experiments. * and **, p < 0.05 and 0.005 compared to the calculated 25 mM starting ethanol concentration respectively; #, p < 0.05 compared to the preceding time point; &, p < 0.05 when comparing middle vs corner wells.

Figure 3.. Ethanol evaporation and effects of compensation with a starting ethanol concentration of 50 mM.

The magnitude of evaporation (black lines) was determined at the indicated timed points following a single bolus addition of a calculated concentration of 50 mM ethanol to the culture wells. The effects of compensation on the ethanol concentrations in the culture wells were evaluated by the addition of 250 mM ethanol at the start (5X Comp, blue lines, +) or 100 and 200 mM ethanol again at the 24 and 48-hour time points, respectively (2X and 4X Comp, red lines, ++) were evaluated. Results are expressed as mean ± SEM from 3 independent experiments. * and ***, p < 0.05 and 0.0005 compared to the calculated 25 mM starting ethanol concentration respectively; #, p < 0.05 compared to the preceding time point; & and &&, p < 0.05 and 0.005, respectively, when comparing middle vs corner wells.

Figure 4.. Ethanol evaporation and effects of compensation with a starting ethanol concentration of 100 mM.

The magnitude of evaporation (black lines) was determined at the indicated timed points following a single bolus addition of a calculated concentration of 100 mM ethanol to the culture wells. The effects of compensation on the ethanol concentrations in the culture wells were evaluated by the addition of 500 mM ethanol at the start (5X Comp, blue lines, +) or 400 mM ethanol again at the 48-hour time point (4X Comp, red lines, ++) were evaluated. Results are expressed as mean ± SEM from 3 independent experiments. *, **, *** and ****, p < 0.05, 0.005, and 0.0005 respectively compared to the calculated 25 mM starting ethanol concentration; #, p < 0.05 compared to the preceding time point; & and &&&, p < 0.05 and 0.0005, respectively, when comparing middle vs corner wells.

Figure 5.. Effect of ethanol evaporation compensation on the viability of neuronal cells.

Human SH-SY5Y dopaminergic neuronal cells were grown to near confluence in 6-well culture plates and treated for 24 hours with 10–100 mM ethanol with and without evaporation compensation. Cell viability was determined with the MTT assay and normalized against that of the media-treated control cells. Results are expressed as mean ± SEM from 5 independent experiments. ** and ****, p < 0.005 and 0.00005 respectively compared to the calculated media (0)-treated control cells; & and &&&, p < 0.05 and 0.0005 respectively no compensation vs with compensation. 0’ indicated the viability of the mock-treated control cells kept in a separate incubator.