Histological analysis of graft survival and immune response of host tissue.

<p>An overview of the mouse brain slice (scale bar: 400 μm) and higher magnification confirm that Iba1 positive cells surround the cell graft (4x magnification, scale bar: 200 μm / 10x magnification, scale bar: 50 μm / 60x magnification, scale bar: 10 μm). GFP-transgene expression (green) and immunostainings with antibodies against: Iba1 (IBA), immunoreaction and HuNu, human nuclei marker. In the lower row 3D images of the IBA staining illustrate the surrounding of the cell graft by the immune cells.</p

<i>In vivo</i>¹⁹F MRI.

<p>(A) High resolution ¹H MR image (left) was acquired as anatomical reference and ¹⁹F MR image (center) was then acquired to localize the implanted cell graft. ¹H and ¹⁹F images were superimposed to combine anatomical information and spatial graft localization (right). (B) Two animals with quantitative depiction of 19F-labelled detectable cells at both two and eight days post implantation. C) Quantification of hNSCs labelled with PFPE at both, day 2 and day 8.</p

Longitudinal evaluation of cell viability by <i>in vivo</i> BLI.

<p>(A) BL images of unlabeled (left) and labeled (right) hNSCs, implanted in the right striatum and longitudinally evaluated from day 0 to day 9 post implantation [¹⁹F labeled cells day 0 (n = 9), day 1 (n = 9), day 2 (n = 8), day 5 (n = 8), day 7 (n = 7), day 9 (n = 5) / unlabeled cells day 0–9 (n = 4)]. (B) SBR (signal to background ratio) normalized to the first time point shows a decrease of cell viability within one week. (+) outliers at least 3x interquartile range.</p

Newly generated hNSCs.

<p>(A) Schematic representation of the designed vector system. The two imaging reporters Luciferase 2 (Luc2) and green fluorescence protein (GFP) are kept under the control of the constitutive active promoter EF1α and are linked via the T2A peptide sequence to ensure equal expression level of the two proteins. (B) Representative microscopic image of transduced and FACS sorted hNSCs. The overlay of the bright-field and fluorescence image is shown right. Scale bar: 50 μm</p

<i>In vitro</i> detectability of ¹⁹F labeled hNSCs by means of ¹⁹F MRI and ¹⁹F MRS.

<p>(A) ¹⁹F MRS of labeled hNSCs and a KF solution as internal standard to quantify the amount of ¹⁹F atoms per cell (B) high resolution ¹H MR image (left), acquired during the same session of the labeled cells ¹⁹F MR image (center). ¹H and ¹⁹F images are then merged to obtain a correct spatial localization (right).</p

Effect of the transduction and ¹⁹F labeling on hNSCs.

<p>(A) Cell viability is shown for WT hNSCs and transgenic EF1-Luc2-GFP hNSCs with and without ¹⁹F labeling (n = 6–8). (B) Cell proliferation was compared among different cell lines. The values were normalized to the WT hNSCs and expressed in percentage (n = 5). (C) <i>In vitro</i> BLI signal from transgenic unlabeled hNSCs compared to ¹⁹F labeled cells. (D) <i>In vitro</i> BLI signal is displayed for a dilution series of cells (labeled and unlabeled) in 6 independent experiments. (+) outliers at least 1.5x interquartile range.</p

Immunohistochemistry validation of grafted hNSCS.

<p>Histology of transplanted H9-EF1-Luc2-GFP cells either labeled with ¹⁹F (n = 4) (A) or unlabeled (n = 4) (B) 9 days after transplantation. An overview of the mouse brain slice (scale bar: 400 μm) and higher magnification of the grafted cells verified the localization of the transplanted cells (4x magnification, scale bar: 200 μm / 10x magnification, scale bar: 50 μm / 60x magnification, scale bar: 10 μm). GFP-transgene expression (green) and immunostainings with antibodies against: DCX, neuronal marker; HuNu, human nuclei marker; Mito, human mitochondria; GFAP, astrocyte marker; Luc, luciferase marker.</p

Histological sections obtained at five weeks after stroke and stained for myelination and inflammation.

<p>ED-1 combined with cresyl violet (A, B, C) shows the presence of macrophage/microglia around the infarct area and in particular between the ventricle and lesion (C); no ED-1 positive cells were observed in the contralesional hemisphere (B). Luxol fast blue (D, E, F) and hematoxylin eosin (G, H, I) staining show the presence of myelin between the ventricle and the infarct (F and I) in contrast to the contralateral side (E and H). GFAP (in green) and CD68 (in red) permitted to detect activated astrocytes and macrophages/microglia, respectively (K) in the border of the lesion area (the asterisk indicates the lesion territory) of the cortex compared to the corresponding contralateral area (J). Comparison of the thickness of the corpus callosum on the ipsilateral and contralteral side (L–O). The luxol fast blue staining of the animal without functional recovery on BOLD shows a distinctly thinner corpus callosum (L,M) compared to the typical situation in a representative case of the other, recovering animals (N,O). Size bar is equivalent to 250 µm in all tissue section overviews (A,D,G,L,M), is 31.25 µm in enlarged sections (B,C,E,F,H,I,M,O) and is 25 µm in the immunohistochemically stained sections (J,K).</p

Tractography in healthy and ischemic conditions.

<p>Tractography of four healthy rat brains (four columns) is performed with corpus callosum as seeding area at two time points (t1, t2) four weeks apart (Panel A). In all cases, the fibers of the corpus callosum followed the white matter going through the internal capsule (yellow arrows), external capsule (white arrows) and fornix (blue arrows). In panel B, tractography in two rats before stroke and one week and four weeks after stroke is depicted. In the two green boxed rows, the reference slice (a gFA map) is close to bregma corresponding to position #2 on the sagittal scheme (green line) whereas in the two red boxed rows, the reference slice is more caudal as indicated by position #1 on the scheme (red line). Lesion volumes, determined from T2WI, are depicted as a yellow mesh. Rat 1 developed a stroke in the caudate putamen, and rat 2 in the caudate putamen and cerebral cortex. The fiber-tracking representation permitted to observe, on the one hand, at 4 weeks after stroke, a modification of the connection between corpus callosum and the internal capsule (white arrows). On the other hand, from one week after stroke, in rats with stroke in caudate-putamen and cerebral cortex (rat 2), an extension of the corpus callosum into the cerebral cortex separating the infarct area and the rest of the cortex (yellow arrows) was observed. The figures show color-coded tracts in the horizontal (red), vertical (green) and transversal (blue) directions, as indicated in the arrow schematic at top right.</p

T₂-weighted coronal images for the evaluation of ischemic territory.

<p>Consecutive T₂-weighted coronal slices (thickness  =  0.5 mm) obtained at two days after stroke at 4.7 T in four representative rats: one rat without edema (A), one with an edema in amygdala (red arrow) (B), one with edema in the caudate putamen (C), and one with edema in the caudate putamen and the cerebral cortex (D).</p