CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.

microRNA-124 (miR-124), the most abundant miRNA of the CNS, was recently shown to modulate the polarization of activated microglia and infiltrating macrophages towards the anti-inflammatory M2 phenotype and protect neurons in various ways after brain disease. In ischemic stroke, microglia and macrophages of a detrimental and persistent pro-inflammatory M1 phenotype have been shown to aggravate the secondary injury. Thus, shifting the polarization of microglia/macrophages into the beneficial, anti-inflammatory M2-like phenotype is considered neuroprotective after stroke onset. Here, we have induced 30 min transient occlusion of the right middle cerebral artery (MCAO) in 34 male, C57BL/6 mice. Lesion development was monitored with T2-weighted MRI. Liposomated miR-124 was injected in 11 animals at 48 h and in 5 animals at 10 days after MCAO. Arg-1, a marker for M2 phenotype, was co-stained with Iba-1, NeuN or GFAP. The distribution of astrocytes, neurons and microglia/macrophages and their expression of Arg-1 were quantified. Early miR-124 injection resulted in a significantly increased neuronal survival and a significantly increased number of M2-like polarized microglia/macrophages. Moreover, the lesion core, delineated by reactive astrocytes, was significantly reduced over time upon early miR-124 injection. These neuroprotective and anti-inflammatory effects of the early miR-124 treatment were pronounced during the first week with Arg-1. Number of Arg-1+ microglia/macrophages correlated with neuronal protection and with functional improvement during the first week. Thus, our present results demonstrate that miR-124 may serve as a novel therapeutic strategy for neuroprotection and functional recovery upon stroke onset.

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.

The inflammatory response following ischemic stroke is dominated by innate immune cells: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.

Background Irreversible damage may occur at reperfusion after sustained cerebral ischaemia. Aims We investigated the value of cyclosporine A for reducing the infarct size in a model of transient middle cerebral artery occlusion. Methods Twenty-seven Sprague-Dawley rats sustained a middle cerebral artery occlusion of one-hour. Acute multimodal Magnetic Resonance Imaging (MRI) was used during occlusion to confirm the success of surgery and measure baseline lesion size. Animals were randomly treated by: (i) intracarotid cyclosporine A (10 mg/kg) 20 mins before middle cerebral artery occlusion (pretreatment group); (ii) intracarotid cyclosporine A (10 mg/kg) immediately after reperfusion (post-treatment group); and (iii) intracarotid saline immediately after reperfusion. Results Histopathological measurements on day 1 showed a significant reduction of infarct size in the pretreatment group compared to the post-treatment (percentage values of ipsilateral hemispheres: 16 ± 5% vs. 29 ± 11%, P= 0·004) and saline groups (16 ± 5% vs. 42 ± 12%, P= 0·015). No significant difference was observed between the post-treatment and saline groups ( P = 0·065). Behavioural examinations on day 1 showed no significant difference between groups. Immunohistochemistry showed a statistically significant reduction of microglial cell count in the pretreatment group compared to either saline or cyclosporine A post-treatment groups. Conclusions We conclude that intracarotid cyclosporine A is effective in reducing infarct size when given prior to ischaemia, but not when administered at reperfusion.

This study sought to evaluate whether the therapeutic effects of an anti-inflammatory drug such as minocycline could be monitored by serial ultrasmall superparamagnetic particles of iron oxide (USPIO)-enhanced MRI in experimental stroke.

The broad aim underlying the present research was to investigate the distribution and homing of bone marrow‐derived macrophages in a rodent model of transient middle cerebral artery occlusion using MRI and ultrasmall superparamagnetic iron oxide (USPIO) to magnetically label bone marrow‐derived macrophages. The specific aim was to assess the intra‐carotid infusion route for bone marrow‐derived macrophage delivery at reperfusion. Fifteen Sprague–Dawley rats sustained 1 h of middle cerebral artery occlusion. USPIO‐labeled bone marrow‐derived macrophages were slowly injected for 5 min immediately after reperfusion in ischemic animals (n = 7), 1 h after the end of surgery in sham animals (n = 5) and very shortly after anesthesia in healthy animals (n = 3). Multiparametric MRI was performed at day 0, just after cell administration, and repeated at day 1. Immunohistological analysis included Prussian blue for iron detection and rat endothelial cell antigen‐1 for endothelium visualization. Intra‐carotid cell delivery brought a large number of cells to the ipsilateral hemisphere of the brain, as seen on both MRI and immunohistology. However, it was associated with high mortality (50%). The study of sham animals demonstrated that intra‐carotid cell delivery could induce ischemic lesions and may thus favor additional brain damage. The present study highlights severe drawbacks to the intra‐carotid delivery of macrophages at the time of reperfusion in this rodent model of transient cerebral ischemia. Multiparametric MRI appears to be a method of choice to monitor longitudinally the effects of cell infusion, allowing the assessment of both cell fate with the help of magnetic labeling and of potential tissue damage. Copyright © 2012 John Wiley & Sons, Ltd.

Epidemiologic studies report cardiovascular protection conferred by omega-3 fatty acids, in particular docosahexaenoic acid (DHA). However, few experimental studies have addressed its potential in acute stroke treatment. The present study used multimodal MRI to assess in vivo the neuroprotection conferred by DHA and by a brain-targeting form of DHA-containing lysophosphatidylcholine (AceDoPC) in experimental stroke. Rats underwent intraluminal middle cerebral artery occlusion (MCAO) and were treated at reperfusion by intravenous injection of i) saline, ii) plasma from donor rats, iii) DHA or iv) AceDoPC, both solubilized in plasma. Twenty-four hours after reperfusion, animals underwent behavioral tests and were sacrificed. Multiparametric MRI (MRA, DWI, PWI, T2-WI) was performed at H0, during occlusion, and at H24, before sacrifice. Brain tissue was used for assay of F(2)-isoprostanes as lipid peroxidation markers. Initial lesion size and PWI/DWI mismatch were comparable in the four groups. Between H0 and H24, lesion size increased in the saline group (mean ± s.d.: +18% ± 20%), was stable in the plasma group (-3% ± 29%), and decreased in the DHA (-17% ± 15%, P=0.001 compared to saline) and AceDoPC (-34% ± 27%, P=0.001 compared to saline) groups. Neuroscores in the AceDoPC group tended to be lower than in the other groups (P=0.07). Treatments (pooled DHA and AceDoPC groups) significantly decreased lipid peroxidation as compared to controls (pooled saline and vehicle) (P=0.03). MRI-based assessment demonstrated the neuroprotective effect of DHA in the MCAO model. Results further highlighted the therapeutic potential of engineered brain-targeting forms of omega-3 fatty acids for acute stroke treatment.

Background Models of intraluminal middle cerebral artery occlusion present an intrinsic variability in infarct size. Behavioral evaluation is frequently performed during arterial occlusion to confirm success of surgery. Aims and/or hypothesis We compared the value of behavioral testing and multimodal magnetic resonance imaging performed during arterial occlusion for identifying successfully operated animals. Methods Rats were tested with behavioral assessment (using three scoring scales and the adhesive removal test) and multimodal magnetic resonance imaging (including magnetic resonance angiography, diffusion-weighted and perfusion-weighted imaging), both performed during the two-hours of middle cerebral artery occlusion using the intraluminal suture model. Behavioral assessment was repeated 24?h after reperfusion, followed by sacrifice. Results Acute apparent diffusion coefficient lesion volume was correlated with both 2,3,5-triphenyl tetrazolium chloride infarct size (r?=?0.75, P?=?0.02) and behavioral status (r?=?0.66, P?=?0.05) on day one. Conversely, no correlation was found between acute behavioral examination and day one outcomes (2,3,5-triphenyl tetrazolium chloride infarct volume, r?=?0.40, P?=?0.28; behavioral examination, r?=?0.39, P?=?0.30). Day zero apparent diffusion coefficient volumes (P?=?0.04), but not behavioral assessment (P?=?0.60), discriminated animals with day one corticostriatal infarcts from these with subcortical infarcts. Conclusions Acute behavioral testing performed during arterial occlusion fails to identify successfully operated animals. Acute diffusion magnetic resonance imaging may be more appropriate to assess and reduce infarct size variability in this model.

To quantify small amounts of iron-labeled cells in mouse brains with magnetic resonance imaging (MRI).