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. 2014 Aug 1;7(10):1193–1203. doi: 10.1242/dmm.015859

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© 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 3. — Comparison of BDNF versus 7,8-DHF, each in soluble and nano formulation, showed effects on rat E14 VM DA neuron survival and maturation. (A) The sets of histograms show % TH⁺ cells in rat E14 VM cultures at 7 days post-plating in differentiation growth medium. In these same cultures, the histograms in B show the average number of primary neurites per DA TH⁺ cell, and C shows the average length of the longest neurite per TH⁺ cell: results for each set are detailed below. Total cell counts of Hoechst-stained nuclei in each culture were made and the effect of therapy on total cell number is stated in the relevant figure legend text. The four treatment groups were BDNF, BDNF-nano, 7,8-DHF and 7,8-DHF-nano: the overall data are summarised in Table 1. The nanoparticles were targeted to Thy-1. No toxicity was observed in any of the culture conditions. (A) BDNF primary E14 VM cells cultured in 50 ng/ml BDNF showed significantly enhanced survival of the DA neurons (one-tailed Student’s t-test, *P<0.05): the total cell count was unchanged (two-tailed Student’s t-test, P>0.05). BDNF-nano primary E14 VM cells cultured in Thy-1-targeted BDNF nanoparticles at 0.1 mg/million cells showed significantly enhanced DA cell survival (one-way ANOVA, F_3,29=3.73, P<0.05, post-hoc Tukey tests, *P<0.05): the total cell count was unchanged (one-way ANOVA, F_3,18=0.54, P>0.05). 7,8-DHF primary E14 VM cells cultured in 7,8-DHF showed a dose-dependent enhancement of DA cell survival at 100 nM and 1 μM (one-way ANOVA, F_4,27=3.65, P<0.05, post-hoc Tukey tests, *P<0.05): treatment had no effect on total cell count (one-way ANOVA, F_4,17=0.28, P>0.05). 7,8-DHF-nano primary E14 VM cells cultured with Thy-1-targeted 7,8-DHF nanoparticles showed significantly enhanced DA cell survival at doses of both 0.05 and 0.1 mg/million cells (one-way ANOVA, F_3,11=8.34, P<0.01, post-hoc Tukey tests, *P<0.05, **P<0.01): treatment had no effect on total cell count at any of the doses tested (one-way ANOVA, F_3,15=0.85, P>0.05). (B,C) BDNF primary E14 VM cells cultured in BDNF showed a significant increase in the average number of primary neurites per DA neuron (one-way ANOVA, F_2,8=62.45, P<0.001, post-hoc Tukey tests, ***P<0.001) and BDNF also increased the average length of the longest neurite (one-way ANOVA, F_2,8=51.87, P<0.001, post-hoc Tukey tests, ***P<0.001). BDNF-nano at 1 mg of Thy-1-targeted BDNF nanoparticles per million cells significantly enhanced the average number of primary neurites per DA neuron (one-way ANOVA, F_3,11=6.63, P<0.05, post-hoc Tukey tests, *P<0.05) and BDNF-nano at this dose significantly increased the length of the longest neurite in DA neurons (one-way ANOVA, F_3,11=7.45, P<0.05, post-hoc Tukey tests, *P<0.05, **P<0.01). 7,8-DHF shows that, although 7,8-DHF had no significant effect on the average number of primary neurites per DA neuron (one-way ANOVA, F_2,7=1.74, P>0.05), 7,8-DHF at 1 μM significantly reduced the average length of the longest neurite (one-way ANOVA, F_2,7=8.85, P<0.05, post-hoc Tukey tests, *P<0.05). 7,8-DHF-nano shows that Thy-1-targeted 7,8-DHF nanoparticles at 0.1 mg per million cells induced a small but significant increase in the average number of primary neurites per DA neuron (one-way ANOVA, F_3,14=3.91, P<0.05, post-hoc Tukey tests, *P<0.05), whereas 7,8-DHF-nano treatment at 1 mg per million cells caused a significant decrease in the average length of the longest primary neurite (one-way ANOVA, F_3,14=18.28, P<0.001, post-hoc Tukey tests, **P<0.01). Quantification was based on five independent repeats (i.e. five litters of embryos) as detailed in the Materials and Methods. (D) Treatment with 50 ng/ml BDNF enhanced maturation of DA neurons in rat E14 VM cultures. Representative images of control and 50 ng/ml BDNF-treated E14 VM cultures at 7 days post-plating in differentiation growth medium with or without 50 ng/ml soluble BDNF. Tyrosine hydroxylase staining is shown in white. Scale bars: 200 μm.