Transplanted Adult Neural Stem Cells Express Sonic Hedgehog In Vivo and Suppress White Matter Neuroinflammation after Experimental Traumatic Brain Injury

Figure 4

Induced genetic in vivo labeling of NSCs expressing Shh after transplantation. Timeline (a) and diagram (b) for host C57BL/6J mice that had TBI or sham surgery and 2 weeks later received an injection into the lateral ventricle (LV) of NSCs isolated from Shh^CreERT2;R26mT/mG mice (NSC-Shh cells). Tamoxifen (TMX) was administered to induce a genetic switch from constitutive mT (red) to mG (green) fluorescence in transplanted NSC-Shh cells expressing Shh. Membranes that were already labeled by constitutive mT and then begin to express mG after TMX may exhibit both mT and mG, so that colabeled membranes appear yellow. Mice were perfused at 4 weeks after TBI or sham surgery. (c) Transplanted NSC-Shh cell distribution within the lateral ventricles and third ventricle. (d) Proportion of transplanted NSC-Shh cells that exhibited expression of mG, indicating in vivo Shh expression. In TBI mice, mG labeling was significantly reduced near the site of injury in the LV but not in the third ventricle. (e–f”) Coronal sections showing transplanted NSC-Shh cells and DAPI nuclear stain (blue) to visualize the choroid plexus (cp) and wall of the LV. Sham mice shown with NSC-Shh cells in the LV that expressed both the mT (red) and mG (green) reporters, with overlap (yellow), indicating in vivo expression of Shh after transplantation (e–e”). TBI mice shown with two clusters of NSC-Shh cells in the LV that express constitutive mT (red) but not Shh-driven mG (green) (f–f”). Shh-driven mG expression was more evident in cells transplanted into the parenchyma, which extended processes ((g–g”), fimbria). After TMX induction, membranes appear green in newly elaborated processes, but appear yellow in cell bodies where mT and mG overlap (arrowheads). Other transplanted cells and processes are labeled by only mT expression (arrows), indicating lack of Shh expression or inefficiency of Cre recombination in vivo. Scale bars = 1 mm (b), 50 μm (d, f), 25 μm (e). ROI = region of interest quantified for designated rostrocaudal extents of the lateral ventricles and the third ventricle (see Methods). Quantification included analysis of a cohort of sham () and TBI () mice.