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K. Xu et al.
lymphatic vessels were capable of carrying hBMSC to the spleen. Indeed, the most novel finding in this study is the discovery that hBMSC were able to migrate directly to the spleen via lymphatic vessels; immunofluorescence analy- sis of the brain and spleen revealed that hBMSC were found within these vessels.
Microglial cells – the resident macrophages of the brain – are activated rapidly in response to brain injury.42,43 Experimental data have shown that resident microglia are activated within minutes of the onset of ischemia and pro- duce a plethora of proinflammatory mediators including interleukin-1β and TNF-a, which exacerbate tissue dam- age44 yet may also protect the brain against ischemic and excitotoxic injury.45,46 Post-ischemic microglial prolifera- tion peaks at 48–72 h after the onset of cerebral ischemia and may last for several weeks after the initial injury.47,48
Given the preferential migration of hBMSC toward microglia under inflammatory conditions seen in vitro, it was hypothesized that the microglia may play a key role in facilitating the transplanted stem cells’ journey into the spleen. Elevated levels of OX6-positive cells were found in both the brain and spleen after stroke, especially in ani- mals with severe stroke. The density of inflammatory cells in the spleen was much higher than in the brain. Greater stroke severity was correlated with a rise in OX6- positive cells and their increased co-localization with
LYVE1 in both the brain and the spleen. The data reveal a more robust migration of OX6-positive cells from the brain to the spleen in response to a more severe stroke. Thus, the present results suggest that elevated inflamma- tion accompanying more severe strokes may also account for the greater successful migration of transplanted stem cells to the spleen. Hence, it is likely that hBMSC possess biodistribution patterns in which they localize around major sites of inflammation, such as the infarct area in the brain and the white pulp in the spleen, which contains numerous inflammatory cells and lymphatic cells that could attract these hBMSC.49 Indeed, high concentrations of hBMSC were deposited in the white pulp of the spleen. We advance the premise that tracking the biodistribution of intracerebrally transplanted hBMSC beyond the spleen, and to other peripheral organs (e.g., thymus) which also mount strong inflammation in response to central nervous system insults, including stroke, represents an innovative paradigm-shifting future investigation (e.g., for brain dis- orders with robust peripheral pathological components, thereby requiring peripheral sequestration of systemic inflammation).
Normally, the BBB functions as an impermeable barri- cade to defend against detrimental pathogens and sub- stances. However, inflammation induced by an ischemic stroke can lead to impaired endothelial activity and com-
AC
B
D
Figure 5. Localization of OX6-positive cells near or within lymphatic vessels in the brain and spleen. (A and B) Staining with OX6, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and DAPI, was performed to visualize microglia/macrophages, lymphatic endothelial cells, and nuclei, respectively, in the brain (A) and spleen (B). Arrow heads indicate co-localization of OX6-positive and LYVE-1-positive cells and show that microglia/macrophages localized near or within lymphatic vessels in the brain and spleen. Pictures taken under 20x and 40x magnification. Scale bars = 100 mm. Red: LYVE-1; green: OX6; blue: DAPI. (A) Images were taken close to the dural sinuses in the brain. (B) Images were taken near the gate of the spleen, close to the white pulp in the spleen. (C and D) In quantitative analyses of the brain (C) and the spleen (D), the stroke groups exhibited higher numbers of co-localized cells than did the sham-treated group (**P<0.01). Significance bars: *P<0.05; **P<0.01; ***P<0.001. (C) a: The group with mild stroke had significantly more co-localization in the brain on day 3 than on other days (P<0.05); b: the group with severe stroke had significantly more co-localized cells in the brain on day 3 than on other days (P<0.01). (D) a: The group with mild stroke had significantly more co-localization in the spleen on day 3 than on other days (P<0.05); b: the group with severe stroke had significantly more co-localized cells in the spleen on day 3 than on other days (P<0.01).
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haematologica | 2019; 104(5)