FIG. 2.

Sema3C embedded in PuraMatrix attracts dopaminergic axonal growth. (A) Scheme of collagen gel coculture, including the ventral Mb explant combined with (i) isthmus (Ist); (ii) pretectum (PT); (iii) hydrogel (Hyd) with Sema3C; (iv) Hyd with control protein (Cp). The rat midbrain (Mb) explants were divided into proximal (P) or distal (D) areas as represented. (B) Mb explant cultured with Ist region. Immunostaining with β III Tubulin and TH shows that dopaminergic axons (DAx) do not grow toward Ist explant. In all cases, the top pictures are reconstructions of low magnification and the images below are enlargements of the boxed areas. (C) Mb explant cultured with PT. DAx grew toward the PT. (D) Mb explant exposed to Hyd with Sema3C. DAx are preferentially present toward the hydrogel. (E) Mb explant cocultured with Hyd Cp. The axons grow randomly. (F) DAx were classified as proximal, distal, or absence of growth and the proportion of attraction, repulsion, or no growth was calculated. PT and Hyd with Sema3C mainly promote dopaminergic attraction; in contrast, Ist and Hyd with Cp mostly induced repulsion. (G) P/D ratios were plotted for the different conditions. PT and Hyd Sema3C promoted attraction, whereas Ist and Hyd Cp conditions induced repulsion. No significant differences were found between PT and Hyd Sema3C. (H) The length of DAx regardless of the direction of growth did not change between all the analyzed conditions. Mean ± standard error of mean *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA and Tukey's multiple comparisons test). Scale bars: 1000 μm (reconstruction images) and 100 μm (magnification images). Color images available online at www.liebertpub.com/tea