Page 218 - 2019_05-HaematologicaMondo-web

K. Xu et al.
ance (ANOVA) followed by a Bonferroni test (GraphPad version 5.01). Comparisons with a P value <0.05 were considered statisti- cally significant.
Results
Infarct sizes
Hematoxylin and eosin staining indicated the presence of infarcts in the animals with mild and severe stroke. Percentages of infarct lesion area were rated accordingly: mild ≤10%, and severe >10% (Figure 1B). The groups with severe stroke had significantly higher percentages of infarct area than those with mild stroke at all time points (P<0.001; P<0.01). Furthermore, transplanting hBMSC grafts did not reduce infarct sizes and no significant out- liers were detected across the treatment groups (Figure 1A).
Survival of human bone marrow mesenchymal stromal cells in the brain and spleen
Confocal microscopy with HuNu staining was used to analyze hBMSC survival and detected positive HuNu expression in brains transplanted with hBMSC (Figure 2A). Additionally, HuNu-positive cells in the spleen indi- cated that intracerebrally injected stem cells migrated from the brain to the spleen (Figure 2B). On day 1, the number of HuNu-positive cells in the brains of the group with severe stroke was significantly higher than that of the group with mild stroke and the sham-treated group (P<0.001). On day 3, the numbers of HuNu-positive cells in the brains of the groups with mild and severe stroke were significantly higher than the number in the sham- treated group P<0.01) (Figure 2C). On all days, the groups with mild and severe stroke had significantly higher num- bers of hBMSC in the spleen compared to the numbers in the spleen of the sham-treated group (P<0.001) (Figure 2D). When comparing between days and within experi- mental groups, there were no significant differences in either the brain or the spleen, between the sham-treated groups (P>0.05) but there were significant differences
between the groups with mild stroke and between the groups with severe stroke (P<0.001) (Figure 2C,D). The detection of surviving hBMSC in the brain and spleen was most robust on day 3, with the highest number of cells identified in the groups with severe stroke (Figure 2C,D).
Human bone marrow mesenchymal stromal cells were visualized within lymphatic vessels in the brain and spleen
To further elucidate whether transplanted stem cells could migrate from the brain to the peripheral immune organs through the lymphatic system, we co-stained for HuNu to visualize hBMSC, and LYVE-1 to visualize lym- phatic endothelial cells. In all animals, few HuNu-positive cells were found within brain lymphatic vessels, which expressed LYVE-1, at any time point (Figure 3A). Additionally, there were no significant differences in the quantity of HuNu and LYVE-1 co-localization in the brain at any time point between sham-treated animals and those with mild or severe stroke (P>0.05) (Figure 3C). In contrast, many hBMSC were visualized within all trans- planted rats’ splenic lymphatic vessels on all days (Figure 3B). Animals with mild and severe strokes displayed sig- nificantly more hBMSC within splenic lymphatic vessels than did sham-treated animals on any day on which measurements were made (P<0.05) (Figure 3D). On day 3, the group with severe stroke expressed the greatest amount of co-localized cells in the spleen (P<0.001).
Stroke induces neuroinflammation in the brain and spleen
To reveal any upregulation of stroke-induced neuroin- flammation, we stained brain and spleen sections with OX6 to label microglia possessing the proinflammatory M1 phenotype (Figure 4A,B). OX6 expression in the brain and spleen was significantly higher in the groups with mild and severe stroke than in the sham-treated group at all time points (P<0.01; P<0.001) (Figure 4C,D). On day 3, the brains and spleens of animals groups with mild and severe stroke, but not those of the sham-treated group, exhibited significantly more OX6-positive cells than they
AB
Figure 1. Infarct sizes in a rat model of stroke. (A) Representative hematoxylin and eosin–stained brain sections from rats that had undergone sham surgery (sham), surgery to induce a mild stroke (mild) or surgery to induce a severe stroke (severe). One hour after sham, mild or severe stroke surgery, rats were intracerebrally injected with human bone marrow mesenchymal stromal cells. Brains from animals with mild or severe stroke show ischemic lesions. Red circles indicate infarct areas. (B) Bar graph depicting infarct size in stroke animals. The infarct area in the ipsilateral hemisphere is expressed as a percentage of the area of the contralateral hemisphere. Values are indicated as means ± standard error of mean. Significance bars: *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.
1064
haematologica | 2019; 104(5)