Neurogenesis upregulation on the healthy hemisphere after stroke enhances compensation for age-dependent decrease of basal neurogenesis.

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.

In Vivo Non-Invasive Tracking of Macrophage Recruitment to Experimental Stroke.

Brain-infiltrating monocyte-derived macrophages are one of the key players in the local immune response after stroke. It is now widely accepted that the inflammatory response is not an exclusively destructive process. However, the underlying molecular mechanisms needed for proper regulation still remain to be elucidated. Here, we propose an in vitro labelling strategy for multimodal in vivo observation of macrophage dynamics distinguished from brain-residing microglia response. Prior to intracerebral transplantation into the striatum of recipient mice or systemic administration, monocytes and macrophages, isolated from luciferase-expressing mice, were labelled with superparamagnetic iron oxide particles. Temporo-spatial localization was monitored by magnetic resonance imaging, whereas survival of grafted cells was investigated using bioluminescence imaging. The labelling procedure of the isolated cells did not significantly influence cell characteristics and resulted in detection of as few as 500 labelled cells in vivo. Two weeks after stereotactic transplantation, the luciferase signal was sustained traceable, with approximately 18% of the original luciferase signal detectable for monocytes and about 30% for macrophages. Hypointensity in MRI of the graft appeared unaltered in spatial location. In a therapeutically relevant approach, systemic cell administration after stroke resulted in accumulation mostly in thoracic regions, as could be visualized with BLI. For detection of homing to ischemic brain tissue more cells need to be administered. Nevertheless, during parallel MRI sessions recruitment of i.v. injected cells to the lesion site could be detected by day 2 post stroke as scattered hypointense signal voids. With further increase in sensitivity, our multi-facetted labelling strategy will provide the basis for in vivo tracking and fate specification of tissue-infiltrating macrophages and their distinct role in stroke-related neuro-inflammation.

Human neural stem cell intracerebral grafts show spontaneous early neuronal differentiation after several weeks.

Human neural stem cells (hNSCs) hold great promise for the treatment of neurological diseases. Considerable progress has been made to induce neural differentiation in the cell culture in vitro and upon transplantation in vivo [2] in order to explore restoration of damaged neuronal circuits. However, in vivo conventional strategies are limited to post mortem analysis. Here, we apply our developed first fate mapping platform to monitor neuronal differentiation in vivo by magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging. Ferritin, Luciferase and GFP under neuronal-specific promoters for immature and mature neurons, respectively, were used to generate transgenic hNSCs. Differentiation-linked imaging reporter expression was validated in vitro. The time profile of spontaneous neuronal maturation after transplantation into mouse brain cortex demonstrated early neuronal differentiation within 6 weeks. Fully mature neurons expressing synaptogenesis were observed only after three months or longer. Our trimodal fate mapping strategy represents a unique non-invasive tool to monitor the time course of neuronal differentiation of transplanted stem cells in vivo.

Limited restoration of visual function after partial optic nerve injury; a time course study using the calcium channel blocker lomerizine.

Secondary degeneration is a process encompassing damage adjacent to a primary injury, usually involving increased Ca(2+) influx into neurons and glia. Lomerizine dihydrochloride is a calcium channel blocker with relatively selective CNS effects, currently in clinical trials for glaucoma. We have recently demonstrated that, following partial transection of the optic nerve (ON), 1 month of lomerizine treatment protects retinal ganglion cells (RGCs), incompletely preserves visual function and also limits elements of secondary degeneration, including macrophage infiltration. However, under some circumstances macrophages have been shown to have different supportive effects on RGC protection and regeneration, casting doubt on the benefit of longer term therapies that reduce macrophage numbers. Here, we determined whether shorter treatment times (1 day or 1 week) result in improved effects on RGC survival and visual function, and whether benefits are maintained after cessation of treatment. We demonstrate that 1 month of lomerizine is the minimum period required to restore the fast reset phase of the optokinetic nystagmus and maintain it for a further 2 months after cessation of treatment (p>0.05, not different from normal). While 1 week of lomerizine treatment results in temporary recovery of numbers of fast reset phases, the recovery is not maintained after treatment cessation. Similarly, protection of RGC densities requires 1 month of lomerizine treatment, but protection is not maintained after treatment cessation. Importantly, none of the lomerizine treatment protocols resulted in full restoration of visual function, confirming the necessity of combining lomerizine with other treatment modalities.