Targeting therapeutic transgene expression to defined tissues is a major task in the development of safe and efficient gene therapy protocols. Recombinant adenovirus is an attractive vector because it can be prepared in huge quantity and new generation vectors possess very large cloning capacities combined with reduced immunogenicity. In the brain, adenovirus transduces mainly glial cells, making it difficult to use this vector system in applications that need expression of therapeutic proteins in neurons. Here, we show that by using a small fragment of the human synapsin 1 gene promoter, we were able to restrict transgene expression from an adenoviral vector exclusively to neurons. Furthermore, we obtained stable long-term transgene expression from this vector in striatum and thalamus at appropriate vector dose. Other promoters like the CMV and U1snRNA promoters also mediated transgene expression over several months, but mainly in glial cells. Although the NSE promoter was relatively neuron specific, it still expressed in glial cells also, and was clearly outperformed by the synapsin promoter with respect to transcriptional neuronal targeting. As an important feature of adenoviral-mediated gene transfer to the brain, we demonstrate that dopaminergic neurons of the substantia nigra do not allow for long-term expression from adenoviral vectors. Strikingly, these neurons appeared to specifically attenuate transgene expression by deleting the adenoviral genome.

Recent evidence suggests that neural stem/precursor cells (NPCs) promote recovery in animal models with delayed neuronal death via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effects is still lacking. Here, we investigated the effects of a delayed transplantation of adult syngenic NPCs—injected intravenously 72 h after transient middle cerebral artery occlusion—on neurological recovery, histopathology and gene expression. NPC-transplanted mice showed a significantly improved recovery from 18 days post-transplantation (dpt) onwards, which persisted throughout the study. A small percentage of injected NPCs accumulated in the brain, integrating mainly in the infarct boundary zone, where most of the NPCs remained undifferentiated up to 30 dpt. Histopathological analysis revealed a hitherto unreported very delayed neuroprotective effect of NPCs, becoming evident at 10 and 30 dpt. Tissue survival was associated with downregulation of markers of inflammation, glial scar formation and neuronal apoptotic death at both mRNA and protein levels. Our data highlight the relevance of very delayed degenerative processes in the stroke brain that are intimately associated with inflammatory and glial responses. These processes may efficaciously be antagonized by (stem) cell-based strategies at time-points far beyond established therapeutic windows for pharmacological neuroprotection.

Therapeutic angiogenesis with vascular endothelial growth factor (VEGF) is a clinically promising strategy in ischaemic disease. The pathophysiological consequences of enhanced vessel formation, however, are poorly understood. We established mice overexpressing human VEGF₁₆₅ under a neuron-specific promoter, which exhibited an increased density of brain vessels under physiological conditions and enhanced angiogenesis after brain ischaemia. Following transient intraluminal middle cerebral artery (MCA) occlusions, VEGF overexpression significantly alleviated neurological deficits and infarct volume, and reduced disseminated neuronal injury and caspase-3 activity, confirming earlier observations that VEGF has neuroprotective properties. Brain swelling was not influenced in VEGF-overexpressing animals, while sodium fluorescein extravasation was moderately increased, suggesting that VEGF induces a mild blood–brain barrier leakage. To elucidate whether enhanced angiogenesis improves regional cerebral blood flow in the ischaemic brain, [¹⁴C]iodoantipyrine autoradiography was performed. Autoradiographies revealed that VEGF induces haemodynamic steal phenomena with reduced blood flow in ischaemic areas and increased flow values only outside the MCA territory. Our data demonstrate that VEGF protects neurons from ischaemic cell death by a direct action rather than by promoting angiogenesis, and suggest that strategies aiming at increasing vascular density in the whole brain, e.g. by VEGF overexpression, may worsen rather than improve cerebral haemodynamics after stroke.

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Systemic transplantation of oxygen−glucose deprivation (OGD)-preconditioned primary microglia enhances neurological recovery in rodent stroke models, albeit the underlying mechanisms have not been sufficiently addressed. Herein, we analyzed whether or not extracellular vesicles (EVs) derived from such microglia are the biological mediators of these observations and which signaling pathways are involved in the process. Exposing bEnd.3 endothelial cells (ECs) and primary cortical neurons to OGD, the impact of EVs from OGD-preconditioned microglia on angiogenesis and neuronal apoptosis by the tube formation assay and TUNEL staining was assessed. Under these conditions, EV treatment stimulated both angiogenesis and tube formation in ECs and repressed neuronal cell injury. Characterizing microglia EVs by means of Western blot analysis and other techniques revealed these EVs to be rich in TGF-β1. The latter turned out to be a key compound for the therapeutic potential of microglia EVs, affecting the Smad2/3 pathway in both ECs and neurons. EV infusion in stroke mice confirmed the aforementioned in vitro results, demonstrating an activation of the TGF-β/Smad2/3 signaling pathway within the ischemic brain. Furthermore, enriched TGF-β1 in EVs secreted from OGD-preconditioned microglia stimulated M2 polarization of residing microglia within the ischemic cerebral environment, which may contribute to a regulation of an early inflammatory response in postischemic hemispheres. These observations are not only interesting from the mechanistic point of view but have an immediate therapeutic implication as well, since stroke mice treated with such EVs displayed a better functional recovery in the behavioral test analyses. Hence, the present findings suggest a new way of action of EVs derived from OGD-preconditioned microglia by regulating the TGF-β/Smad2/3 pathway in order to promote tissue regeneration and neurological recovery in stroke mice.