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. 2015 Feb 17;18(5):pyu090. doi: 10.1093/ijnp/pyu090

Figure 2.

Figure 2.

DCEF effects on neuronal elements. N2a mouse cells were differentiated into neuronal-like cells and various aspects relating to viability (A), morphology (B), and response (C) were assessed following exposure to increasing DCEFs (0, 25, 50, and 100V/m). (A) The total percentage of viable N2a cells (as measured in 3–4 replicates, n total=877–1900 cells) as well as the percentage of viable N2a cells specifically depicting neurites (5–6 replicates, n total=901–1134) was similar across all DCEFs applied (from 0 to 100V/m). (C) Protein concentration for GAP-43, a molecule expressed during axonal/neurite growth, was increased following a 24-hour stimulation of 25 and 50V/m (graph depicting average data of 4 replicates). (D) Measurements of total neurite length revealed significant elongation (5–6 replicates, n total=449–624) (E) as well as a shift of orientation towards the cathode (5–6 replicates, n total=449–624) when cells were stimulated at 25, 50, and 100V/m. The polar distribution of neurite angles (each dot represents the angle of a single neurite) demonstrates a significantly more prominent orientation towards the cathode at 50 and 100V/m. (F) Additionally, the 25 and 50 V/m DCEFs had a significant effect on the total length of the cell body (5–6 replicates, n total=493–688) when compared with cells exposed to 0 or 100V/m DCEF. (G) Polar distribution of cell body angles (each dot represents the angle of a single cell body) reveals that cell angles reorient parallel to the DCEF. (H-K) Photomicrographs of double phalloidin (F-actin, red) and DAPI (nucleus, blue) immunofluorescent staining illustrating the morphological and orientational changes in N2a cells in response to DCEFs (scale bar: 20 µm). (A-D, F) The values represent LS-means ± SEM. Statistical analyses were performed using ANOVA (split-plot-type) (A-D, F). (E) Kuiper’s Test of Uniformity and (G) chi-square test using grouped data (number of classes = 8 with identical divisions for interval 0–180°). ***P<.001 vs 0V/m; ## P<.01, ### P<.001 vs 25V/m; & P<.05, &&& P<.001 vs 50V/m. DCEF, direct current electrical field; GAP-43, growth associated protein-43; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LS, least square; OD, optical density; V/m, Volt/meter.