Intravascular delivery of NSC is a promising experimental treatment strategy for ischemic stroke. We have previously shown that NSC express the integrin CD49d, potentially allowing them to actively enter the ischemic brain from the vascular compartment. Enrichment of NSC for CD49d improved this process and resulted in better functional recovery.8
Here, we suggest that CCL2 is a crucial molecule governing transendothelial recruitment of NSC in the central nervous system. CCL2, a member of the C-C motif subfamily of chemokines,17
is known to promote chemotaxis of monocytes and hematopoietic progenitors18–21
to sites of inflammation in the brain. In experimental models of stroke, CCL2 is upregulated in the ischemic areas;22–24
in stroke patients, elevated CCL2 levels have been found in the systemic circulation.25,26
CCL2 accumulates in the perivascular space of the cerebral microvasculature, is actively relayed across the blood– brain barrier, and is presented on the luminal plasma membrane of endothelial cells, where it acts as a chemoattractant factor for leukocytes.27
These lines of evidence suggest that CCL2 may be significantly involved in the recruitment of intra-arterially delivered CCR2-expressing NSC across the blood– brain barrier.
Intraparenchymal migration of NSC in response to CCL2 has been described.13,28,29
However, CCL2 may act through receptors other than CCR2.30
Using an in vitro migration assay, we found that CCR2+/+
NSC exhibited a dose-dependent migratory response to CCL2 whereas CCR2−/−
NSC did not. To rule out that CCR2−/−
NSC may have lost their general migratory capacity, we demonstrated chemotaxis in response to CXCR12a, which acts on the receptor CXCR4.31
These data support the hypothesis that the observed effects are specifically mediated by the CCL2/CCR2 interaction.
In response to ischemic stroke in rodents, CCL2 is significantly upregulated in the brain after 6 hours, peaks at 24 hours, and gradually declines to baseline levels over the next 5 days.13
Thus, we chose the time point of 24 hours after stroke for transplantation. After intracarotid delivery, we observed significantly higher numbers of CCR2+/+
NSC recruited into the ischemic brain areas as compared to CCR2−/−
cells, proving the importance of CCR2 for active homing of NSC across the blood– brain barrier (). Accordingly, we observed improved locomotor recovery in animals that received CCR2+/+
grafts as compared to mice transplanted with CCR2−/−
NSC (). To demonstrate dependence on CCL2 in a converse experiment, we transplanted CCR2-expressing NSC into WT and CCL2 knockout animals. This experiment further confirmed the necessity of the CCL2/CCR2 interaction for transendothelial recruitment of NSC to the injured brain (Supplemental Figure II
Previous studies have reported that CCL2 deficiency attenuates infarct volumes in focal rodent stroke models,32,33
which could represent a confounding factor. However, we found only a minor difference in lesion size on T2-weighted MRI 72 hours after stroke in CCL2+/+
animals using the HI model (Supplemental Figure IIB
), which would not be sufficient to explain the difference in the number of recruited WT NSC between CCL2+/+
To overcome the limitations of histology in assessing whole brain distribution and dynamic changes over time in the same subject, we monitored the dynamics of NSC recruitment to the stroke brain areas during the early period after intra-arterial delivery using BLI of luciferase-transfected CCR2+/+ and CCR2−/− NSC. We found that the majority of the CCR2+/+ cells were specifically recruited to the hemisphere with lesions during the first few hours, whereas CCR2−/− NSC showed almost no homing (). Analysis of the left–right redistribution of the BLI signal in both CCR2+/+ and CCR2−/− animals (, left/right ratio) revealed a possible secondary recruitment of NSC to the brain from the systemic circulation. Cell death on the ipsilateral side could also account for the observed changes in left/right ratio. Whereas the CCR2/CCL2 interaction is crucial for recruitment of NSC, the lineage determination fate was not dependent on the CCR2 receptor status of the transplanted NSC in our experiments ().
Because it allows reperfusion after ischemia, the HI model of stroke offers advantages for the study of intravascular stem cell delivery techniques as compared to focal ischemia models based on permanent vessel occlusion. In the present study, HI resulted in reproducible cortico-striatal infarction on the ipsilateral side of the brain. Because differentiated oligodendrocytes are less susceptible to ischemic damage than oligodendrocyte progenitors, white matter affection is less pronounced in adult mice than in the neonatal HI model.
We further investigated the relation of transmigrated NSC and the cerebral vasculature. NSC that undergo transendothelial migration also may directly participate in angiogenesis. A recent article demonstrated that PDGFR-β
–positive pericytes were required for blood– brain barrier integrity during embryogenesis.34
It also has been shown that circulating cells expressing PDGFR-β
were crucial for the maturation of brain vessels after stroke.35
We found that 12% of our transplanted GFP-positive NPC were positive for PDGFR-β
Histology and BLI data suggest that intra-arterially delivered NSC need to cross the blood– brain barrier to improve behavioral recovery. This finding is in line with our previous study investigating the effects of vascular cell adhesion molecule-1/CD49d.8
Other groups, however, have demonstrated that mesenchymal stem cells do not necessarily need to enter the brain for improving recovery from neurological deficits,36,37
and immunomodulatory and/or neuroprotective mechanisms have been postulated to mediate the observed effects on behavioral outcome. However, to date, no such data are available for NSC or neural progenitors, and it remains to be elucidated whether homing to the ischemic brain, enabling neural differentiation and facilitating delivery of secreted growth factors and cytokines, also might have additional beneficial effects for mesenchymal stem cells. Several mechanisms have been implicated in cell-mediated functional recovery. The proangiogenic vascular endothelial growth factor-mediated role of transplanted stem cells has been extensively studied.38
Here, we demonstrate an immunomodulatory mechanism with reduction in CD68-positive cells and a neuroprotecitve effect with reduction in Fluoro-Jade–positive cells () and reduction in lesion size. All these mechanisms are potentially involved in stem cells-mediated recovery.