Figure 6

The role of secondary signaling messengers in constructive remodeling of esophageal tissue defects. (a) Immunoblot analysis of Akt target, p-mTOR and β-tubulin protein levels from neotissues treated with vehicle or Akt inhibitor for 7 days of scaffold implantation. Vehicle and inhibitor comparisons were made on the same immunoblots and with equal amounts of protein loaded per lane (N = 3–4 animals per group). Targeted bands were cropped from original full length immunoblots displayed in Supplementary Fig. S2. (b,c) Representative images of global and magnified esophageal cross-sections from neotissues (NT) treated with vehicles or RTK inhibitors with or without pan-caspase inhibition for 7 days post-op. Specimens were stained with Masson’s trichrome (top 2 rows in each panel) or subjected to immunohistochemical analysis for pan-cytokeratin (CK) expression (bottom row in each panel). Scale bars for top, middle, and bottom rows are respectively 1.25 mm, 500 μm, and 200 μm. (e) Immunohistochemical evaluations of Ki67 + pan-CK + epithelial cells at the host-graft interface in specimens described in (b,c). Scale bars in (h) for all photomicrographs are 200 μm. For (b–h), respective pan-CK expression is displayed in red, Ki67 expression is displayed in green and blue represents DAPI nuclear counterstain. Scale bars in (e) for all photomicrographs are 200 μm. For (b,c,e), respective pan-CK expression is displayed in red, Ki67 expression is displayed in green and blue represents DAPI nuclear counterstain. (d,f) Histomorphometric evaluations of pan-CK expression in NSC, untreated controls and neotissues described in (b,c) as well as the percentage of Ki67 + pan-CK + epithelial cells at the host-graft interface in samples described in (e). N = 4–8 rats per group and 2–4 sections per animal were analyzed for each data point. For panels (d,f), multi-group (> 3 groups) comparisons were performed with the Kruskal Wallis test in combination with the post hoc Dunn’s test. Two group comparisons were performed with the Mann–Whitney U test. (*) = p < 0.05 in comparison to respective vehicle control. (#) = p < 0.05 in comparison to respective inhibitor group. (β) = p < 0.05 in comparison to NSC. Data are expressed as means ± standard deviations.