Ischemic stroke and cerebral infarction triggered by the blockage of blood supply can cause damage to the brain via a complex series of pathological changes. Recently, diverse therapies have emerged as promising candidates for the treatment of stroke. These treatments exert therapeutic effects by acting on diverse target molecules and cells in different time windows from the acute to chronic phases. Here, using immunohistochemistry, we show pathophysiological changes in the brain microenvironment at the hyperacute (within 6 h), acute (1~3 days), subacute (7 days), and chronic (1 month) phases following ischemic injury. The progression of damage to the brain was evaluated by immunohistochemistry for NeuN+ neurons, GFAP+ astrocytes, and Iba1+ microglia, and by the emergence of the cell death-related molecules such as AIF, FAF1, and Cleaved caspase-3. In our previous study, we showed that the transplantation of neural-induced mesenchymal stem cells by introducing Neurogenin1 (MSC/Ngn1) dramatically improved the stroke outcome compared to the parental MSCs in an acute stroke rat model. Here, we investigated the therapeutic effects of intra-arterial transplantation of MSC/Ngn1 cells in a chronic stroke model. Mannitol, hyperosmotic agent, used for opening the blood-brain barrier (BBB) to facilitate the infiltration of transplanted cells toward the infarcted area in a chronic stroke model. First, we identified that treatment of mannitol increased degradation of tight junction proteins, such as ZO-1 and claudin-5, and BBB permeability with measuring transendothelial electrical resistance (TEER) and diffusion of 150kDa FITC-dextran in vitro. Additionally, BBB permeability was evaluated with Evans blue dye extravasation into the brain parenchyma of chronic ischemic stroke model. Osmotic opening of BBB by intra-arterial infusion of mannitol was effective for 1 hour in both of normal and chronic stroke brain. Administering pre-treatment of mannitol has facilitated transmigration of MSC and MSC/Ngn1 through BBB in both in vitro trans-well migration assay and in vivo cell transplantation. MSCs or MSCs/Ngn1 were intra-arterial (IA) injected in the chronic stroke model after IA mannitol pre-treatment. The cells were labeled with superparamagnetic iron oxide (SPIO)-nanoparticles for magnetic resonance imaging (MRI) cell tracking. Significantly higher number of SPIO-labeled cells were in the mannitol-treated group compared with saline-treated group. MRI analysis revealed that mannitol increased SPIO-labeled MSCs and MSCs/Ngn1 delivery to ipsilateral hemisphere of chronic stroke rat model. To detect and quantify administered cells in the brain, human AluJ quantitative polymerase chain reaction (qPCR) analysis was performed. And we examined the rats with immunohistological analysis and behavioral tests. Transmigrated MSCs/Ngn1 were differentiated neuronal cells and mannitol-pretreated rats showed significantly promoted neurological functional recovery. These results indicated that mannitol facilitated the extravasation of stem cells and increased BBB permeability before intra-arterial transplantation has been shown to be even more effective in cell therapy of chronic stroke injury.
