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Detrimental Impact of Energy Drink Compounds on Developing Oligodendrocytes and Neurons

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Detrimental Impact of Energy Drink Compounds on Developing Oligodendrocytes and Neurons

Meray Serdar et al. Cells.

Abstract

The consumption of energy drinks is continuously rising, particularly in children and adolescents. While risks for adverse health effects, like arrhythmia, have been described, effects on neural cells remain elusive. Considering that neurodevelopmental processes like myelination and neuronal network formation peak in childhood and adolescence we hypothesized that developing oligodendrocytes and neurons are particularly vulnerable to main energy drink components. Immature oligodendrocytes and hippocampal neurons were isolated from P0-P1 Wistar rats and were incubated with 0.3 mg/mL caffeine and 4 mg/mL taurine alone or in combination for 24 h. Analysis was performed immediately after treatment or after additional three days under differentiating conditions for oligodendrocytes and standard culture for neurons. Oligodendrocyte degeneration, proliferation, and differentiation were assessed via immunocytochemistry and immunoblotting. Neuronal integrity was investigated following immunocytochemistry by analysis of dendrite outgrowth and axonal morphology. Caffeine and taurine induced an increased degeneration and inhibited proliferation of immature oligodendrocytes accompanied by a decreased differentiation capacity. Moreover, dendritic branching and axonal integrity of hippocampal neurons were negatively affected by caffeine and taurine treatment. The negative impact of caffeine and taurine on developing oligodendrocytes and disturbed neuronal morphology indicates a high risk for disturbed neurodevelopment in children and adolescents by excessive energy drink consumption.

Keywords: caffeine; energy drinks; neuron; oligodendrocytes; taurine.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Caffeine and taurine alone or in combination induce immature oligodendrocyte cell loss. After three days under standard culture conditions primary immature oligodendrocytes were incubated with or without 0.3 mg/mL caffeine, 4 mg/mL taurine, and the combination of both for 24 h. (A) Representative images of immunofluorescence staining of immature oligodendrocytes (A2B5 green, Olig2 red). (B) Quantification of Olig2 positive cells. The density of immature oligodendrocytes was determined by measuring the A2B5 positive area (C). Data are derived from 15 images per group out of three independent experiments (depicted by circle = control, square = caffeine, triangle up = taurine, triangle down = caffeine + taurine). Scale bar 400 µm. *** p < 0.001, one-way ANOVA followed by Bonferroni post hoc test.
Figure 2
Figure 2
Immature oligodendrocyte cell loss is caused by a decreased proliferation and increased degeneration induced by caffeine and taurine. After treatment with or without 0.3 mg/mL caffeine and 4 mg/mL taurine alone or in combination, cells were analysed for proliferation via immunocytochemistry and for apoptosis via western blot. (A) Representative images of immunofluorescence staining of proliferating oligodendrocytes (PCNA green, Olig2 red). The number of proliferating oligodendrocytes was quantified in 15 images per group derived from three independent experiments (B) Scale bar 400 µm. Ratio of cCaspase-3 and GAPDH protein expression was analyzed in protein lysates of cultured oligodendrocytes derived from four independent experiments (C), depicted by circle = control, square = caffeine, triangle up = taurine, triangle down = caffeine + taurine). *p < 0.05, *** p < 0.001, one-way ANOVA followed by a Bonferroni post hoc test.
Figure 3
Figure 3
Myelination capacity of immature oligodendrocytes is decreased by caffeine and taurine. Primary immature oligodendrocytes were treated with or without 0.3 mg/mL caffeine, 4 mg/mL taurine, and the combination of both for 24 h followed by media exchange to differentiating conditions for another three days. (A) Representative images of immunofluorescence staining for the pan-oligodendrocyte marker Olig2 (red) and the myelination marker MBP (green) to visualize mature oligodendrocytes. The density of oligodendrocytes was quantified by counting Olig2 positive cells (B). MBP-positive oligodendrocytes were quantified by subtracting MBP-negative cells from the total Olig2-positive cell number. (C). Data are derived from 15 images per group out of three independent experiments, depicted by circle = control, square = caffeine, triangle up = taurine, triangle down = caffeine + taurine. Scale bar 400 µm. ** p < 0.01, *** p < 0.001, one-way ANOVA followed by a Bonferroni post hoc test.
Figure 4
Figure 4
Caffeine and taurine alone or in combination reduce branching of dendrites in primary hippocampal neurons. Following three days in culture primary hippocampal neurons were incubated with or without 0.3 mg/mL caffeine, 4 mg/mL taurine and the combination of both for 24 h. (A) Dendrites were visualized by immunofluorescence staining for microtubule-associated protein 2 (Map2, green). Morphological changes were measured by defining primary dendrites (A, bottom row, red) and higher dendrites (A, bottom row, blue). The amount of total (B), primary (C), and higher (D) dendrites was quantified; subfigures (EG) represent results of quantification of dendritic lengths, respectively. Data are derived from 25–30 images per group out of three independent experiments (n = 83–103 cells per group, depicted by circle = control, square = caffeine, triangle up = taurine, triangle down = caffeine + taurine). Scale bar 100 µm. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by a Bonferroni post hoc test.
Figure 5
Figure 5
Caffeine and taurine lead to sustained reduced dendritic branching and disturbed axonal network formation. After three days in culture primary hippocampal neurons were treated with or without 0.3 mg/mL caffeine, 4 mg/mL taurine and the combination of both for 24 h followed by cultivation under standard culture conditions for another three days. (A) Representative images of Map2 (green) stained dendrites. Quantifications of the amount of total, primary and higher dendrites, as well as their lengths are shown in subfigures (BG). Data are derived from 25–30 images per group out of three independent experiments (n = 75–82 cells per group, depicted by circle = control, square = caffeine, triangle up = taurine, triangle down = caffeine + taurine). Morphological changes of axons were visualized by immunofluorescence staining for TAU (H). Scale bar 100 µm. *** p < 0.001, one-way ANOVA followed by a Bonferroni post hoc test.

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