Thrombolysis remains the only beneficial therapy for ischemic stroke, but is restricted to a short therapeutic window following the infarct. Currently research is focusing on spontaneous regenerative processes during the sub-acute and chronic phase. Angiogenesis, the formation of new blood vessels from pre-existing ones, was observed in stroke patients, correlates with longer survival and positively affects the formation of new neurons. Angiogenesis takes place in the border zones of the infarct, but further insight into the temporal profile is needed to fully apprehend its therapeutic potential and its relevance for neurogenesis and functional recovery. Angiogenesis is a multistep process, involving extracellular matrix degradation, endothelial cell proliferation, and, finally, new vessel formation. Interaction between vascular endothelial growth factor and its receptor 2 (VEGFR2) plays a central role in these angiogenic signaling cascades. In the present study we investigated non-invasively the dynamics of VEGFR2 expression following cerebral ischemia in a mouse model of middle cerebral artery occlusion (MCAO). We used a transgenic mouse expressing firefly luciferase under the control of the VEGFR2 promotor to non-invasively elucidate the temporal profile of VEGFR2 expression after stroke as a biomarker for VEGF/VEGFR2 signaling. We measured each animal repetitively up to 2 weeks after stroke and found increased VEGFR2 expression starting 3 days after the insult with peak values at 7 days. These were paralleled by increased VEGFR2 protein levels and increased vascular volume in peri-infarct areas at 14 days after the infarct, indicating that signaling via VEGFR2 leads to successful vascular remodeling. This study describes VEGFR2-related signaling is active at least up to 2 weeks after the infarct and results in increased vascular volume. Further, this study presents a novel strategy for the non-invasive evaluation of angiogenesis-based therapies.

After stroke, the brain has shown to be able to achieve spontaneous functional recovery despite severe cerebral damage. This phenomenon is poorly understood. To address this issue, focal transient ischemia was induced by 60 min middle cerebral artery occlusion in Wistar rats. The evolution of stroke was followed using two magnetic resonance imaging modalities: diffusion spectrum imaging (acquired before, one and four weeks after stroke) and functional magnetic resonance imaging (acquired before and five weeks after stroke). To confirm the imaging observations, immunohistochemical staining for myelin, astrocytes and macrophages/microglia was added. At four weeks after stroke, a focal alteration of the diffusion anisotropy was observed between the ipsilesional ventricle and the lesion area. Using tractography this perturbation was identified as reorganization of the ipsilesional internal capsule. Functional imaging at five weeks after ischemia demonstrated activation of the primary sensorimotor cortex in both hemispheres in all rats except one animal lacking a functional response in the ipsilesional cortex. Furthermore, fiber tracking showed a transhemispheric fiber connection through the corpus callosum, which-in the rat without functional recovery-was lost. Our study shows the influence of the internal capsule reorganization, combined with inter-hemispheric connections though the corpus callosum, on the functional activation of the brain from stroke. In conclusion, tractography opens a new door to non-invasively investigate the structural correlates of lack of functional recovery after stroke.

Intravenous administration of iron oxide nanoparticles after experimental stroke has been shown to produce focal signal intensity changes in the ischemic boundary on MRI images. These changes have been attributed to the influx of iron-laden blood-borne macrophages, although it has been suggested that this effect might not always be completely specific to inflammatory cells. The aim of the present study was to investigate this phenomenon in a subacute time frame that is more relevant to the peripheral inflammatory response. Imaging experiments (T(2) -, T(2)*- and T(1) -weighted sequences) were acquired in Wistar rats 6 days after transient middle cerebral artery occlusion (MCAO). Animals were intravenously infused with different doses of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (300, 600 or 1000 µmol Fe/kg), or saline and gadolinium, and imaged again 24 h later. Tissue was immediately processed for immunohistochemistry with the macrophage marker ED-1, in combination with Prussian blue for iron. Ischemic tissue exhibited a large increase in T(2) values, and overall contrast enhancement was apparent in the brain and surrounding muscle. In contrast with previous reports, there were no regions of focal signal intensity changes in the ischemic territory in any of the images, although a region of interest analysis revealed a trend towards iron accumulation in the ischemic hemisphere, particularly in the cortex of T(2)* images. However, histological examination revealed that, despite extensive ED-1-positive macrophage accumulation in the entire ischemic territory, none of these cells were Prussian blue positive, except in the meninges of one animal that received a high dose of USPIO nanoparticles. These results imply that the observed trend is a result of the presence of contrast agent in the blood, or meninges, and not iron-containing inflammatory cells.

Stroke is a leading cause of death and disability worldwide with no treatment for the chronic phase available. Interestingly, an endogenous repair program comprising inflammation and neurogenesis is known to modulate stroke outcome. Several studies have shown that neurogenesis decreases with age but the therapeutic importance of endogenous neurogenesis for recovery from cerebral diseases has been indicated as its ablation leads to stroke aggravation and worsened outcome. A detailed characterization of the neurogenic response after stroke related to ageing would help to develop novel and targeted therapies. In an innovative approach, we used the DCX-Luc mouse, a transgenic model expressing luciferase in doublecortin-positive neuroblasts, to monitor the neurogenic response following middle cerebral artery occlusion over three weeks in three age groups (2, 6, 12months) by optical imaging while the stroke lesion was monitored by quantitative MRI. The individual longitudinal and noninvasive time profiles provided exclusive insight into age-dependent decrease in basal neurogenesis and neurogenic upregulation in response to stroke which are not accessible by conventional BrdU-based measures of cell proliferation. For cortico-striatal strokes the maximal upregulation occurred at 4days post stroke followed by a continuous decrease to basal levels by three weeks post stroke. Older animals effectively compensated for reduced basal neurogenesis by an enhanced sensitivity to the cerebral lesion, resulting in upregulated neurogenesis levels approaching those measured in young mice. In middle aged and older mice, but not in the youngest ones, additional upregulation of neurogenesis was observed in the contralateral healthy hemisphere. This further substantiates the increased propensity of older brains to respond to lesion situation. Our results clearly support the therapeutic relevance of endogenous neurogenesis for stroke recovery and particularly in older brains.

We generated transgenic human neural stem cells (hNSCs) stably expressing the reporter genes Luciferase for bioluminescence imaging (BLI) and GFP for fluorescence imaging, for multimodal imaging investigations. These transgenic hNSCs were further labeled with a clinically approved perfluoropolyether to perform parallel 19F MRI studies. In vitro validation demonstrated normal cell proliferation and differentiation of the transgenic and additionally labeled hNSCs, closely the same as the wild type cell line, making them suitable for in vivo application. Labeled and unlabeled transgenic hNSCs were implanted into the striatum of mouse brain. The time profile of their cell fate after intracerebral grafting was monitored during nine days following implantation with our multimodal imaging approach, assessing both functional and anatomical condition. The 19F MRI demarcated the graft location and permitted to estimate the cell number in the graft. BLI showed a pronounce cell loss during this monitoring period, indicated by the decrease of the viability signal. The in vivo obtained cell fate results were further validated and confirmed by immunohistochemistry. We could show that the surviving cells of the graft continued to differentiate into early neurons, while the severe cell loss could be explained by an inflammatory reaction to the graft, showing the graft being surrounded by activated microglia and macrophages. These results are different from earlier cell survival studies of our group where we had implanted the identical cells into the same mouse strain but in the cortex and not in the striatum. The cortical transplanted cells did not show any loss in viability but only pronounced and continuous neuronal differentiation.

The stroke model of distal middle cerebral artery occlusion is considered a reliable stroke model with high reproducibility and low mortality rate. Thus, it is preferred for assessments of therapeutic strategies, in particular for neurorepair and regeneration studies. However, present literature has reported only on the lesion behavior and behavioral deficits during the acute and subacute phase of maximally three weeks. We have here aimed to characterize the lesion expansion and consequent, potential tissue displacements using structural magnetic resonance imaging modalities, histology, and behavioral tests, during the chronic time window of 12 weeks following stroke induction. We found a severe cortical thinning resulting in 15% tissue loss of the ipsilateral cortex by 6 weeks. After two weeks, massive hippocampus displacement was found, into the cortical tissue void and, in this process, pushing the corpus callosum to the brain surface showing an almost radial direction towards the surface. These massive chronic morphological changes and rearrangements, not known from other stroke models, have relevant consequences for decision of stem cell graft placement for cerebral regeneration to assure persistent graft vitality during a longitudinal investigation in the chronic phase.

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Transplantation of neural stem cells (NSCs) appears to be a promising regenerative therapy for a variety of neurological disorders. Nevertheless, NSC engraftment is limited by the number of surviving cells. To maximize stem cell-mediated effects, timing of implantation and cell number have to be precisely evaluated. Here, a transgenic murine NSC line was optimized for high expression levels of the imaging reporters Luc2 and copGFP. NSCs of 150 000, 75 000, 15 000 or 1500 cells or Hanks buffered salt solution were implanted into the striatum of nude mice. The survival of NSCs was monitored with in vivo bioluminescence imaging (BLI) over 2 weeks and brain sections were histologically analysed for glial cells of the innate immune system. The longitudinal in vivo BLI data revealed a significantly reduced viability with the highest rate for 150 000 engrafted NSCs. The cell loss was not correlated with the number of Iba-1 immune cells nor GFAP astrocytes. Histological quantification of copGFP cells at 14 days postimplantation confirmed the in vivo data with the highest density of copGFP cells in the 150 000-cell graft and the highest survival rate for 1500 cells/graft. In conclusion, regenerative therapies should strictly evaluate the maximal number of stem cells to be transplanted in one location, as the results suggest that there is a critical limit of cells able to survive in the adult brain. Survival is limited by availability of oxygen and nutrients but not the inflammatory response induced by the implantation.