Josephine Herz

Extracellular Vesicles Improve Post-Stroke Neuroregeneration and Prevent Postischemic Immunosuppression

Although the initial concepts of stem cell therapy aimed at replacing lost tissue, more recent evidence has suggested that stem and progenitor cells alike promote postischemic neurological recovery by secreted factors that restore the injured brains capacity to reshape. Specifically, extracellular vesicles (EVs) derived from stem cells such as exosomes have recently been suggested to mediate restorative stem cell effects. In order to define whether EVs indeed improve postischemic neurological impairment and brain remodeling, we systematically compared the effects of mesenchymal stem cell (MSC)‐derived EVs (MSC‐EVs) with MSCs that were i.v. delivered to mice on days 1, 3, and 5 (MSC‐EVs) or on day 1 (MSCs) after focal cerebral ischemia in C57BL6 mice. For as long as 28 days after stroke, motor coordination deficits, histological brain injury, immune responses in the peripheral blood and brain, and cerebral angiogenesis and neurogenesis were analyzed. Improved neurological impairment and long‐term neuroprotection associated with enhanced angioneurogenesis were noticed in stroke mice receiving EVs from two different bone marrow‐derived MSC lineages. MSC‐EV administration closely resembled responses to MSCs and persisted throughout the observation period. Although cerebral immune cell infiltration was not affected by MSC‐EVs, postischemic immunosuppression (i.e., B‐cell, natural killer cell, and T‐cell lymphopenia) was attenuated in the peripheral blood at 6 days after ischemia, providing an appropriate external milieu for successful brain remodeling. Because MSC‐EVs have recently been shown to be apparently safe in humans, the present study provides clinically relevant evidence warranting rapid proof‐of‐concept studies in stroke patients.

Very-late-antigen-4 (VLA-4)-mediated brain invasion by neutrophils leads to interactions with microglia, increased ischemic injury and impaired behavior in experimental stroke

AbstractNeuronal injury from ischemic stroke nis aggravated by invading peripheral immune cells. Early infiltrates of neutrophil granulocytes and T-cells influence the outcome of stroke. So far, however, neither the timing nor the cellular dynamics of neutrophil entry, its consequences for the invaded brain area, or the relative importance of T-cells has been extensively studied in an intravital setting. Here, we have used intravital two-photon microscopy to document neutrophils and brain-resident microglia in mice after induction of experimental stroke. We demonstrated that neutrophils immediately rolled, firmly adhered, and transmigrated at sites of endothelial activation in stroke-affected brain areas. The ensuing neutrophil invasion was associated with local blood–brain barrier breakdown and infarct formation. Brain-resident microglia recognized both endothelial damage and neutrophil invasion. In a cooperative manner, they formed cytoplasmic processes to physically shield activated endothelia and trap infiltrating neutrophils. Interestingly, the systemic blockade of very-late-antigen-4 immediately and very effectively inhibited the endothelial interaction and brain entry of neutrophils. This treatment thereby strongly reduced the ischemic tissue injury and effectively protected the mice from stroke-associated behavioral impairment. Behavioral preservation was also equally well achieved with the antibody-mediated depletion of myeloid cells or specifically neutrophils. In contrast, T-cell depletion more effectively reduced the infarct volume without improving the behavioral performance. Thus, neutrophil invasion of the ischemic brain is rapid, massive, and a key mediator of functional impairment, while peripheral T-cells promote brain damage. Acutely depleting T-cells and inhibiting brain infiltration of neutrophils might, therefore, be a powerful early stroke treatment.

Role of Neutrophils in Exacerbation of Brain Injury After Focal Cerebral Ischemia in Hyperlipidemic Mice

Background and Purpose— Inflammation-related comorbidities contribute to stroke-induced immune responses and brain damage. We previously showed that hyperlipidemia exacerbates ischemic brain injury, which is associated with elevated peripheral and cerebral granulocyte numbers. Herein, we evaluate the contribution of neutrophils to the exacerbation of ischemic brain injury. Methods— Wild-type mice fed with a normal chow and ApoE knockout mice fed with a high cholesterol diet were exposed to middle cerebral artery occlusion. CXCR2 was blocked using the selective antagonist SB225002 (2 mg/kg) or neutralizing CXCR2 antiserum. Neutrophils were depleted using an anti-Ly6G antibody. At 72 hours post ischemia, immunohistochemistry, flow cytometry, and real-time polymerase chain reaction were performed to determine cerebral tissue injury and immunologic changes in the blood, bone marrow, and brain. Functional outcome was assessed by accelerated rota rod and tight rope tests at 4, 7, and 14 days post ischemia. Results— CXCR2 antagonization reduced neurological deficits and infarct volumes that were exacerbated in hyperlipidemic ApoE−/− mice. This effect was mimicked by neutrophil depletion. Cerebral neutrophil infiltration and peripheral neutrophilia, which were increased on ischemia in hyperlipidemia, were attenuated by CXCR2 antagonization. This downscaling of neutrophil responses was associated with increased neutrophil apoptosis and reduced levels of CXCR2, inducible nitric oxide synthase, and NADPH oxidase 2 expression on bone marrow neutrophils. Conclusions— Our data demonstrate a role of neutrophils in the exacerbation of ischemic brain injury induced by hyperlipidemia. Accordingly, CXCR2 blockade, which prevents neutrophil recruitment into the brain, might be an effective option for stroke treatment in patients with hyperlipidemia.

Transduction of Neural Precursor Cells with TAT‐Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

Novel therapeutic concepts against cerebral ischemia focus on cell‐based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell‐based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein‐transduced with TAT‐heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT‐Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT‐Hsp70‐transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT‐Hsp70‐transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain‐derived neurotrophic factor, glial cell line‐derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood‐brain‐barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long‐term neuroprotection. STEM CELLS2012;30:1297–1310

Intracerebroventricularly delivered VEGF promotes contralesional corticorubral plasticity after focal cerebral ischemia via mechanisms involving anti-inflammatory actions

Vascular endothelial growth factor (VEGF) has potent angiogenic and neuroprotective effects in the ischemic brain. Its effect on axonal plasticity and neurological recovery in the post-acute stroke phase was unknown. Using behavioral tests combined with anterograde tract tracing studies and with immunohistochemical and molecular biological experiments, we examined effects of a delayed i.c.v. delivery of recombinant human VEGF(165), starting 3 days after stroke, on functional neurological recovery, corticorubral plasticity and inflammatory brain responses in mice submitted to 30 min of middle cerebral artery occlusion. We herein show that the slowly progressive functional improvements of motor grip strength and coordination, which are induced by VEGF, are accompanied by enhanced sprouting of contralesional corticorubral fibres that branched off the pyramidal tract in order to cross the midline and innervate the ipsilesional parvocellular red nucleus. Infiltrates of CD45+ leukocytes were noticed in the ischemic striatum of vehicle-treated mice that closely corresponded to areas exhibiting Iba-1+ activated microglia. VEGF attenuated the CD45+ leukocyte infiltrates at 14 but not 30 days post ischemia and diminished the microglial activation. Notably, the VEGF-induced anti-inflammatory effect of VEGF was associated with a downregulation of a broad set of inflammatory cytokines and chemokines in both brain hemispheres. These data suggest a link between VEGFs immunosuppressive and plasticity-promoting actions that may be important for successful brain remodeling. Accordingly, growth factors with anti-inflammatory action may be promising therapeutics in the post-acute stroke phase.

The novel proteasome inhibitor BSc2118 protects against cerebral ischaemia through HIF1A accumulation and enhanced angioneurogenesis

Only a minority of stroke patients receive thrombolytic therapy. Therefore, new therapeutic strategies focusing on neuroprotection are under review, among which, inhibition of the proteasome is attractive, as it affects multiple cellular pathways. As proteasome inhibitors like bortezomib have severe side effects, we applied the novel proteasome inhibitor BSc2118, which is putatively better tolerated, and analysed its therapeutic potential in a mouse model of cerebral ischaemia. Stroke was induced in male C57BL/6 mice using the intraluminal middle cerebral artery occlusion model. BSc2118 was intrastriatally injected 12 h post-stroke in mice that had received normal saline or recombinant tissue-plasminogen activator injections during early reperfusion. Brain injury, behavioural tests, western blotting, MMP9 zymography and analysis of angioneurogenesis were performed for up to 3 months post-stroke. Single injections of BSc2118 induced long-term neuroprotection, reduced functional impairment, stabilized blood-brain barrier through decreased MMP9 activity and enhanced angioneurogenesis when given no later than 12 h post-stroke. On the contrary, recombinant tissue-plasminogen activator enhanced brain injury, which was reversed by BSc2118. Protein expression of the transcription factor HIF1A was significantly increased in saline-treated and recombinant tissue-plasminogen activator-treated mice after BSc2118 application. In contrast, knock-down of HIF1A using small interfering RNA constructs or application of the HIF1A inhibitor YC1 (now known as RNA-binding motif, single-stranded-interacting protein 1 (RBMS1)) reversed BSc2118-induced neuroprotection. Noteworthy, loss of neuroprotection after combined treatment with BSc2118 and YC1 in recombinant tissue-plasminogen activator-treated animals was in the same order as in saline-treated mice, i.e. reduction of recombinant tissue-plasminogen activator toxicity through BSc2118 did not solely depend on HIF1A. Thus, the proteasome inhibitor BSc2118 is a promising new candidate for stroke therapy, which may in addition alleviate recombinant tissue-plasminogen activator-induced brain toxicity.

Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury

OBJECTIVEnPreterm brain injury is a major cause of disability in later life, and may result in motor, cognitive and behavioural impairment for which no treatment is currently available. The aetiology is considered as multifactorial, and one underlying key player is inflammation leading to white and grey matter injury. Extracellular vesicles secreted by mesenchymal stem/stromal cells (MSC-EVs) have shown therapeutic potential in regenerative medicine. Here, we investigated the effects of MSC-EV treatment on brain microstructure and maturation, inflammatory processes and long-time outcome in a rodent model of inflammation-induced brain injury.nnnMETHODSn3-Day-old Wistar rats (P3) were intraperitoneally injected with 0.25mg/kg lipopolysaccharide or saline and treated with two repetitive doses of 1×108 cell equivalents of MSC-EVs per kg bodyweight. Cellular degeneration and reactive gliosis at P5 and myelination at P11 were evaluated by immunohistochemistry and western blot. Long-term cognitive and motor function was assessed by behavioural testing. Diffusion tensor imaging at P125 evaluated long-term microstructural white matter alterations.nnnRESULTSnMSC-EV treatment significantly ameliorated inflammation-induced neuronal cellular degeneration reduced microgliosis and prevented reactive astrogliosis. Short-term myelination deficits and long-term microstructural abnormalities of the white matter were restored by MSC-EV administration. Morphological effects of MSC-EV treatment resulted in improved long-lasting cognitive functions INTERPRETATION: MSC-EVs ameliorate inflammation-induced cellular damage in a rat model of preterm brain injury. MSC-EVs may serve as a novel therapeutic option by prevention of neuronal cell death, restoration of white matter microstructure, reduction of gliosis and long-term functional improvement.

Visualization of macroscopic cerebral vessel anatomy—A new and reliable technique in mice

Visualizing rodent cerebral vasculature is an important tool in experimental stroke research. Intravascular perfusion with colored latex has been the method of choice until recently. However, latex perfusion has some technical limitations which compromise its reproducibility. We therefore describe a simple and reproducible method to visualize cerebral vessels in mice. A mixture of two commercially available carbon black inks is injected into the thoracic aorta resulting in efficient filling and high contrast visualization of cerebral vessels. Feasibility of this technique has been validated by identifying anastomotic points between anterior and middle cerebral arteries. Furthermore, perfusion with combined carbon inks allows visualization of significantly smaller vessel diameters at a higher vessel density in comparison to perfusion with diluted/undiluted latex. Thus, perfusion with combined carbon inks offers a simple, cost-effective and reproducible technique in order to visualize cerebral vasculature.

Interaction between hypothermia and delayed mesenchymal stem cell therapy in neonatal hypoxic-ischemic brain injury

Acute hypothermia treatment (HT) is the only clinically established intervention following neonatal hypoxic-ischemic brain injury. However, almost half of all cooled infants still die or suffer from long-lasting neurological impairments. Regenerative therapies, such as mesenchymal stem cells (MSC) appear promising as adjuvant therapy. In the present study, we hypothesized that HT combined with delayed MSC therapy results in augmented protection, improving long-term neurological outcome. Postnatal day 9 (P9) C57BL/6 mice were exposed to hypoxia-ischemia followed by 4u202fh HT. Murine bone marrow-derived MSC (1u202f×u202f106u202fcells/animal) were administered intranasally at P12. Cytokine and growth factor levels were assessed by ELISA and Luminex® multiplex assay 24u202fh following MSC delivery. One week after HI, tissue injury and neuroinflammatory responses were determined by immunohistochemistry and western blot. Long-term motor-cognitive outcome was assessed 5u202fweeks post injury. MSC responses to the brains environment were evaluated by gene expression analysis in MSC, co-cultured with brain homogenates isolated at P12. Both, MSC and HT improved motor deficits, while cognitive function could only be restored by MSC. Compared to each single therapy, combined treatment led to increased long-lasting motor-cognitive deficits and exacerbated brain injury, accompanied by enhanced endothelial activation and peripheral immune cell infiltration. MSC co-cultured with brain extracts of HT-treated animals revealed increased pro-inflammatory cytokine and decreased growth factor expression. In vivo protein analysis showed higher pro-inflammatory cytokine levels after combined treatment compared to single therapy. Furthermore, HI-induced increase in growth factors was normalized to control levels by HT and MSC single therapy, while the combination induced a further decline below control levels. Our results suggest that alteration of the brains microenvironment by acute HT modulates MSC function resulting in a pro-inflammatory environment combined with alteration of the homeostatic growth factor milieu in the neonatal hypoxic-ischemic brain. This study delineates potential unexpected side effects of cell-based therapies as add-on therapy for acute hypothermia treatment.

Intravascular perfusion of carbon black ink allows reliable visualization of cerebral vessels.

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes (i.e. carmine red, India ink) or latex with or without carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen. Maeda et al. have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.