6 research outputs found

    hADSCs promote axonal regeneration.

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    <p>A–D) Photomontages showing the expression pattern of 5HT-positive serotonergic fibers in horizontal sections spanning the pre- and post-lesion regions in DMEM (A) and hADSC (B) treated animals one week after compression. The inset shows an example of a hogback termination in the post-lesion area. Note that the hADSC-treated cord exhibits a larger number of serotoninergic fibers crossing the injury site. C) Graph representing the total length of 5-HT fibers in each experimental group (n = 3 in each group). D–E) 5HT expression was greatly reduced in the chronic phase (8 wks) in DMEM-treated rats (D), while it was slightly reduced in hADSC (E) treated group. F) Quantification of the total length of 5-HT positive axons, eight weeks after SCI (n = 3 in each group). G–R) Progression of axonal regeneration was followed by Tuj1 (G–L) or anti-GAP-43 (M–R) immunoreactivity. Panels on the left show Tuj1 (G, J) or GAP-43 (M, P) positive fibers one week after compression at the epicenter of the lesion. Panels H, K, N, Q and rows on the right show respective rostral (left), epicenter (center), and caudal (right) regions of the same section eight weeks after SCI. Far column on the right (yellow frame) shows boxed areas in higher magnification. The dashed line indicates the border of the lesion cavity. Graphics show the quantification of the total lengths of Tuj1 (I, L) or GAP-43 (O, R) positive fibers one week (I, O) and eight weeks (L, R) after SCI in the peri-cavity region (n = 3 in each group). Tuj1 and GAP-43 positive axons in DMEM (blue) and hADSC (red) treated groups were quantified in an area of approximately 900 µm in extension along the longitudinal axis. Both white and grey matter was analyzed. The y-axes in panels F, I, L, O and R were omitted for aesthetic purpose and they represent fiber length per area (µm/µm<sup>2</sup>×10<sup>3</sup>). The values in C, F, I, L, O, R represent means ± standard errors. *P<0.05, **P<0.01, ***P<0.001. Bars: A, B = 500 µm, D–Q = 100 µm, high magnification = 50 µm.</p

    hADSCs led to the appearance of perivascular spaces in between endothelial and astrocytic basement membranes one week after injection.

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    <p>A–D) Confocal images of horizontal sections immunostained with anti-pan-laminin antibody (red) one week after injury. Note that in DMEM animals (A,B) there is no separation between the two membranes whereas in hADSCs–treated animals (C,D) these membranes are separated (arrows in D). E) Confocal images of a horizontal section immunostained with anti-pan-laminin (green) and RECA-1(red). F–F”) Confocal imagens of sequential optical sections immunostained with anti-pan-laminin (green) and DAPI (blue) showing the extravasation of cells from the blood vessels. Bars: C, F = 50 µm B, D, E = 25 µm.</p

    hADSCs induce functional recovery and reduce cavitation and cellular inflammatory response.

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    <p>The locomotor performance was assessed by the BBB score. Animals were evaluated weekly, during eight weeks after spinal compression (A). Asterisks indicate statistic differences between hADSC (n = 9) and DMEM groups (n = 9) and crosses indicate differences between hADSC and sham groups (n = 3). B,C) Low-magnification photomontages showing the marked difference in distribution of macrophages in the spinal cord of rats treated with hADSC (C) and DMEM (B). Note that ED1-positive cells concentrate in the immediate surroundings of the lesion cavity in hADSC-treated animals (arrowheads), in contrast to the dispersed distribution in DMEM-treated animals. D, E) GFAP immunoreactivity delineates the cavity and reveals astrocyte activation following injury, in vehicle (DMEM) (D) and hADSC (E) treated animals. F) Graph representing the area of the lesion cavity of 5 different dorso–ventral sections (represented in the x axis) in DMEM (blue) and hADSC (red) treated animals (n = 3 per condition). Note that cavitation in hADSC-treated animals is smaller. G,H) High magnification images of the boxed areas in <i>D</i> (blue) and <i>E</i> (red) are shown in <i>G</i> and H, respectively. Note the few GFAP-positive cells at the vicinity of the lesion and that very few cells are seen at the cavity border in hADSC-treated rat (H). I) Graph representing the total area of positive staining for GFAP (see <i>Materials and Methods</i>) in the immediate surroundings of the cavity in DMEM (blue) and hADSC (red) treated animals (n = 3 per condition). Dashed line delineates the cavity borders in G and H. *P<0.05, **P<0.01, ***: p<0.001. Bars: B and C = 500 µm, D and E = 100 µm and G and H = 50 µm.</p

    Laminin produced by hADSC forms reticular-like deposits independent of fractones.

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    <p>Photomontage of a horizontal section of the undamaged spinal cord one week after injection of hADSCs (A) immunostained with a pan-specific anti-laminin antibody (green) and (B) counterstained with DAPI (blue). The arrowheads in B delimit the region of the highest increase in cellularity, corresponding to reticular laminin in A. Boxed areas in A were amplified to show the detailed morphology of the types of laminin deposits in the spinal cord. C) The red box shows fractones characterized by the presence of thin lines (arrow) and puncta (arrowhead). D) The yellow box shows an independent reticular-like deposit. E) The blue box shows laminin around a blood vessel. Note that laminin seems to detach from the perivascular basal lamina and to spread in the spinal parenchyma (asterisks). Panels C and D were reconstructed in the z axis to provide a 3D view of the fractones (F) and of the reticular deposits (G). In F, lines (arrow) and puncta (arrowheads) presented similar shapes in 2D or 3D. Panel H shows a 3D view of laminin deposits stained with an antibody specific for the α2 chain of human laminin (red) and counterstained with DAPI (blue). The 3D structure of laminin secreted by hADSCs is similar to the one depicted in G and different from the one depicted in F. Note that DAPI-stained nuclei are nested within reticular laminin (red) and that the protein produced by GFP-transduced hADSCs (green) largely surpasses the borders of the secreting cells.</p

    hADSCs secrete laminin in the spinal cord independently of SCI.

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    <p>A–C) Horizontal section of an animal subjected to spinal compression and injected with GFP-hADSCs one week after injury/injection. Panels A and B show photomontages depicting DAPI staining (A) and immunoreactivity for anti-human laminin (B). Note that cell infiltrates and laminin are located in corresponding regions. Panel C shows confocal images demonstrating the coincident localization of GFP-positive hADSCs (green), cell infiltrates (blue) and human laminin (red). D–E) Horizontal section of an uninjured animal one week after transplantation with GFP-hADSCs. Panels D to D’’’ show photomontages depicting DAPI staining (blue, D), GFP-positive hADSCs (D’, green), immunoreactivity for anti-human laminin (D’’, white) and immunoreactivity for anti-pan laminin (D’’’, red). Panel E shows confocal images to demonstrate that while the anti-pan laminin antibody labels rat blood vessels (E’’, red), the anti-human laminin does not (E’). Bars: A, B, D = 1 mm, C = 100 µm, E = 200 µm.</p

    Areas of increased cellularity induced by hADSC are mainly composed of neural precursors, but cell types differ in the vascular and parenchymal compartments.

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    <p>A–F) Confocal images of horizontal sections of the spinal cord showing immunostaining for nestin (A), vimentin (B), NG2 (C), smooth muscle α-actin (αSMA, D), Tuj1 (E), and Olig2 (F) in clusters of high cell density along the midline, one week after SCI. Note that cells in the clusters are positive for all of these markers, suggesting that clusters are mostly composed of neural progenitors and possibly of invading pericytes. Panels on the right correspond to high-magnification images of the boxed areas, showing the labeling for each phenotypic marker. G–M) Blood vessels immunostained with pan-laminin (red) and nestin (G), vimentin (H), NG2 (I), αSMA (J), Tuj1 (K) (green). Panels L and M show that most Olig2 positive cells (purple) are out of blood vessels (L). Panel M depicts one Olig2-labeled nucleus in close proximity with a region where the basal lamina seems to be disrupted (arrow). (N–Q) Correspondence between immunoreactivities for nestin (N) and Ki67 (O) in photomontages of horizontal sections of an injured spinal cord one week after injury/injection. Confocal images showing proliferative activity (anti-Ki67, red) in the spinal parenchyma (P) and a perivascular region (Q) of nestin positive cells. Bars: A–F = 100 µm; G–M = 50 µm; N, O = 1 mm, Q = 10 µm.</p
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