Histological and ultrastructural comparison of cauterization and thrombosis stroke models in immune-deficient mice

<p>Abstract</p> <p>Background</p> <p>Stroke models are essential tools in experimental stroke. Although several models of stroke have been developed in a variety of animals, with the development of transgenic mice there is the need to develop a reliable and reproducible stroke model in mice, which mimics as close as possible human stroke.</p> <p>Methods</p> <p>BALB/Ca-RAG2^-/-γc^-/-mice were subjected to cauterization or thrombosis stroke model and sacrificed at different time points (48hr, 1wk, 2wk and 4wk) after stroke. Mice received BrdU to estimate activation of cell proliferation in the SVZ. Brains were processed for immunohistochemical and EM.</p> <p>Results</p> <p>In both stroke models, after inflammation the same glial scar formation process and damage evolution takes place. After stroke, necrotic tissue is progressively removed, and healthy tissue is preserved from injury through the glial scar formation. Cauterization stroke model produced unspecific damage, was less efficient and the infarct was less homogeneous compared to thrombosis infarct. Finally, thrombosis stroke model produces activation of SVZ proliferation.</p> <p>Conclusions</p> <p>Our results provide an exhaustive analysis of the histopathological changes (inflammation, necrosis, tissue remodeling, scarring...) that occur after stroke in the ischemic boundary zone, which are of key importance for the final stroke outcome. This analysis would allow evaluating how different therapies would affect wound and regeneration. Moreover, this stroke model in RAG 2^-/-γC ^-/-allows cell transplant from different species, even human, to be analyzed.</p

Transplantation of hMSCs and hMAPCs induced an increase in angiogenesis.

<p>A) CD31 immunostaining in the ischemic boundary zone at different time points after cell transplantation (2 days, 4 days, 7 days and 28 days) (Scale bar = 100 µm). B) Quantitative analysis of angiogenesis after cell transplantation (n = 4). *<i>p</i><0.05 and ** <i>p</i><0.001 by Bonferroni test.</p

Glial-scar inhibitory effect after hMSCs and hMAPCs transplantation.

<p>Immunoflurescence analysis of fibronectin (A) and NG2-CSP (B) 4 days after hMSCs and hMAPCs transplantation. The intensity of the staining varied according to the color scale shown in the left corner in each panel. C) Quantitative analysis of NG2-CSP immunostaining. *<i>P</i><0.01 in PBS-treated group vs cell transplanted groups. Scale bar = 100 µm.</p

Therapeutic Effects of hMAPC and hMSC Transplantation after Stroke in Mice

<div><p>Stroke represents an attractive target for stem cell therapy. Although different types of cells have been employed in animal models, a direct comparison between cell sources has not been performed. The aim of our study was to assess the effect of human multipotent adult progenitor cells (hMAPCs) and human mesenchymal stem cells (hMSCs) on endogenous neurogenesis, angiogenesis and inflammation following stroke. BALB/Ca-RAG 2^−/− γC^−/− mice subjected to FeCl₃ thrombosis mediated stroke were intracranially injected with 2×10⁵ hMAPCs or hMSCs 2 days after stroke and followed for up to 28 days. We could not detect long-term engraftment of either cell population. However, in comparison with PBS-treated animals, hMSC and hMAPC grafted animals demonstrated significantly decreased loss of brain tissue. This was associated with increased angiogenesis, diminished inflammation and a glial-scar inhibitory effect. Moreover, enhanced proliferation of cells in the subventricular zone (SVZ) and survival of newly generated neuroblasts was observed. Interestingly, these neuroprotective effects were more pronounced in the group of animals treated with hMAPCs in comparison with hMSCs. Our results establish cell therapy with hMAPCs and hMSCs as a promising strategy for the treatment of stroke.</p> </div

Effect of hMSCs and hMAPCs transplantation on microglia activation after stroke.

<p>A) Immunostaining with anti-Iba1 in sections corresponding to the peri-infarct zone 2 days after cell transplantation. Upper panel represents a panoramic view (Scale bar = 50 µm) while a detail image is provided in the lower panel (Scale bar = 10 µm). B) Quantification of the mean number of intersections of microglial processes/cell with the grid. C) Representation of the percentage of intersections with each concentric circle of the grid/cell. *<i>p</i><0.05 for PBS-treated group vs. hMAPCs by Bonferroni test.</p

Effect of transplantation of hMSCs and hMAPCs in tissue loss.

<p>A) Nissl-staining photomicrographs of coronal sections that show the loss of tissue at 28 days after transplantation. The dashed line marks tissue loss. Scale bar = 1000 µm; B) Quantification of tissue loss 28 days after cell transplantation n = 4 *<i>p</i><0.05 PBS-treated group vs cell transplanted groups by Bonferroni test.</p

Endogenous neuroblast survival.

<p>A–C) Double immunostaining against DCX (green), HuNu (red) and DAPI (blue) in the peri-infarct zone 28 days after cell tranplantation. Scale bar = 50 µm. Magnification scale bar = 75 µm. D) Representation of the DCX/DAPI index estimated in the scar boundary zone 28 days after hMSCs and hMAPCs transplantation. *<i>p</i><0.05 for PBS-treated group vs hMAPCs by Bonferroni test.</p