Delayed tissue plasminogen activator (tPA) treatment increases the risk of intracerebral hemorrhage in patients with ischemic stroke. We previously demonstrated that tPA treatment caused hemorrhagic complications in a 4-h middle cerebral artery occlusion (MCAO) mouse model when administered after reperfusion. In the present study, we administered an anti-high mobility group box 1 (αHMGB1) antibody to 4-h MCAO mice to evaluate the usability of αHMGB1 antibody treatment in the delayed phase of ischemia, beyond the therapeutic time window of tPA.αHMGB1 antibody, tPA and control IgG were dissolved in normal saline and administered intravenously into the tail vein of the mice after reperfusion. Infarct volume, hemorrhagic volume, brain swelling, functional outcomes and levels of pro-inflammatory cytokines, such as HMGB1, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, were evaluated 24 h after MCAO.tPA treatment was not only ineffective but also caused a massive intracerebral hemorrhage. Treatment with αHMGB1 antibody reduced the infarct volume and swelling and ameliorated neurologic impairment and motor coordination without hemorrhagic complications by inhibiting HMGB1 activity. Moreover, the αHMGB1 antibody suppressed pathways of secondary inflammatory responses, such as IL-6 and TNF-α, after cerebral ischemia.These results indicate that αHMGB1 antibody may be therapeutically efficient in the delayed phase of ischemia, where tPA treatment is no longer an eligible option. Treatment with an αHMGB1 antibody may be an effective therapeutic option in patients who exceed the tPA therapeutic time window.
Endothelial progenitor cells (EPCs) have been extensively investigated as a therapeutic approach for repairing the vascular system in cerebrovascular diseases. Beyond vascular regeneration per se, EPCs may also release factors that affect the entire neurovascular unit. Here, we aim to study the effects of the EPC secretome on oligovascular remodeling in a mouse model of white matter injury after prolonged cerebral hypoperfusion.The secretome of mouse EPCs was analyzed with a proteome array. In vitro, the effects of the EPC secretome and its factor angiogenin were assessed on primary oligodendrocyte precursor cells and mature human cerebral microvascular endothelial cells (hCMED/D3). In vivo, mice were subjected to permanent bilateral common carotid artery stenosis, then treated with EPC secretome at 24 hours and at 1 week, and cognitive outcome was evaluated with the Y maze test together with oligodendrocyte precursor cell proliferation/differentiation and vascular density in white matter at 4 weeks.Multiple growth factors, cytokines, and proteases were identified in the EPC secretome, including angiogenin. In vitro, the EPC secretome significantly enhanced endothelial and oligodendrocyte precursor cell proliferation and potentiated oligodendrocyte precursor cell maturation. Angiogenin was proved to be a key factor since pharmacological blockade of angiogenin signaling negated the positive effects of the EPC secretome. In vivo, treatment with the EPC secretome increased vascular density, myelin, and mature oligodendrocytes in white matter and rescued cognitive function in the mouse hypoperfusion model.Factors secreted by EPCs may ameliorate white matter damage in the brain by boosting oligovascular remodeling.
Holes and grooves of a few tens of micrometers have been fabricated on glass plates by femtosecond laser pulses in air. Shapes and surface morphology of the fabricated structures have been studied in detail by making their replicas. Details of the internal shape of the strictures formed have been studied by making replicas of them. Development of the shape of drilled hole with the increase in irradiated pulse number shows some distinguishable stages. Smaller number of pulses make channel at the center of rather flat bottom. With the increase of pulse number, the channel changed into conical, funnel-like shape as the depth increased. Shape of the groove depends on the scanning speed, i.e., number of irradiated pulses on the same position. The groove show little cracking or beaking with clear edges. Some amount of debris deposited along the groove but these debris were easily removed by smear. Inner surface of the groove has coarser morphology than that of the hole. Depth of the grove was shallower than that of the hole, even though the number of irradiated pulse was estimated to be equal.
Abstract Die Diels‐Alder‐Reaktion der im Titel genannten Verbindung (I) mit den cyclischen Dienen (II) und (V) führt selektiv zu den Addukten (III) und (IV) bzw. (VI) mit exocyclischer Allylsulfon‐Gruppe.
Reactive astrocytes are traditionally thought to impede brain plasticity after stroke. However, we previously showed that reactive astrocytes may also contribute to stroke recovery, partly via the release of a nuclear protein called high-mobility group box 1 (HMGB1). Here, we investigate the mechanisms that allow stimulated astrocytes to release HMGB1. Exposure of rat primary astrocytes to IL-1beta for 24 h elicited a dose-dependent HMGB1 response. Immunostaining and western blots of cell lysates showed increased intracellular levels of HMGB1. Western blots confirmed that IL-1beta induced a release of HMGB1 into astrocyte conditioned media. MAP kinase signaling was involved. Levels of phospho-ERK were increased by IL-1beta, and the MEK/ERK inhibitor U0126 decreased HMGB1 upregulation in the stimulated astrocytes. Since HMGB1 is a nuclear protein, the role of the nuclear protein exporter, chromosome region maintenance 1 (CRM1), was assessed as a candidate mechanism for linking MAP kinase signaling to HMGB1 release. IL-1beta increased CRM1 expression in concert with a translocation of HMGB1 from nucleus into cytoplasm. Blockade of IL-1beta-stimulated HMGB1 release with the ERK inhibitor U0126 was accompanied by a downregulation of CRM1. Our findings reveal that IL-1beta stimulates the release of HMGB1 from activated astrocytes via ERK MAP kinase and CRM1 signaling. These data suggest a novel pathway by which inflammatory cytokines may enhance the ability of reactive astrocytes to release prorecovery mediators after stroke.
Although VEGF165 (vascular endothelial growth factor-165) is able to enhance both angiogenesis and neurogenesis, it also increases vascular permeability through the blood-brain barrier. Heparan sulfate (HS) sugars play important roles in regulating VEGF bioactivity in the pericellular compartment. Here we asked whether an affinity-purified VEGF165-binding HS (HS7) could augment endogenous VEGF activity during stroke recovery without affecting blood-brain barrier function.Both rat brain endothelial cell line 4 and primary rat neural progenitor cells were used to evaluate the potential angiogenic and neurogenic effects of HS7 in vitro. For in vivo experiments, male Sprague-Dawley rats were subjected to 100 minutes of transient focal cerebral ischemia, then treated after 4 days with either PBS or HS7. One week later, infarct volume, behavioral sequelae, immunohistochemical markers of angiogenesis and neural stem cell proliferation were assessed.HS7 significantly enhanced VEGF165-mediated angiogenesis in rat brain endothelial cell line 4 brain endothelial cells, and increased the proliferation and differentiation of primary neural progenitor cells, both via the VEGFR2 (vascular endothelial growth factor receptor 2) pathway. Intracerebroventricular injection of HS7 improved neurological outcome in ischemic rats without changing infarct volumes. Immunostaining of the compromised cerebrum demonstrated increases in collagen IV/Ki67 and nestin/Ki67 after HS7 exposure, consistent with its ability to promote angiogenesis and neurogenesis, without compromising blood-brain barrier integrity.A VEGF-activating glycosaminoglycan sugar, by itself, is able to enhance endogenous VEGF165 activity during the post-ischemic recovery phase of stroke.
Based on an experimental model of warfarin-associated intracerebral hemorrhage, we investigated whether the rapid reversal of anticoagulation using prothrombin complex concentrates (PCC) or recombinant activated coagulation factor VII (rFVIIa) reduces hematoma volume.Mice were orally pretreated with warfarin (2 mg/kg). Intracerebral hemorrhage was induced by collagenase injection into the right striatum. Forty-five minutes later, PCC (100 IE/kg), rFVIIa (1 mg/kg), or an equal volume of saline was administered intravenously. Hematoma volume after 24 hours was quantified using a photometric hemoglobin assay.International normalized ratio was 4.3±0.4 in saline-treated mice, 0.9±0.1 in rFVIIa mice, and 1.4±0.2 in PCC mice. Intracerebral hemorrhage volume was 29.0±19.7 μL in the saline group (n=7), 8.6±4.3 μL in the rFVIIa group (n=6), and 6.1±1.8 μL in the PCC group (n=7; analysis of variance between-group differences P=0.004; post hoc rFVIIa versus saline P=0.021; PCC versus saline P=0.007). No significant difference was found between PCC- and rFVIIa-treated animals.Our results suggest that PCC and rFVIIa are equally effective in restoring coagulation and preventing excessive hematoma growth in acute warfarin-associated intracerebral hemorrhage.
Abstract Background Transplantation of mitochondria is increasingly explored as a novel therapy in central nervous system (CNS) injury and disease. However, there are limitations in safety and efficacy because mitochondria are vulnerable in extracellular environments and damaged mitochondria can induce unfavorable danger signals. Methods Mitochondrial O-GlcNAc-modification was amplified by recombinant O-GlcNAc transferase (OGT) and UDP-GlcNAc. O-GlcNAcylated mitochondrial proteins were identified by mass spectrometry and the antiglycation ability of O-GlcNAcylated DJ1 was determined by loss-of-function via mutagenesis. Therapeutic efficacy of O-GlcNAcylated mitochondria was assessed in a mouse model of transient focal cerebral ischemia-reperfusion. To explore translational potential, we evaluated O-GlcNAcylated DJ1 in CSF collected from patients with subarachnoid hemorrhagic stroke (SAH). Results We show that isolated mitochondria are susceptible to advanced glycation end product (AGE) modification, and these glycated mitochondria induce the receptor for advanced glycation end product (RAGE)-mediated autophagy and oxidative stress when transferred into neurons. However, modifying mitochondria with O-GlcNAcylation counteracts glycation, diminishes RAGE-mediated effects, and improves viability of mitochondria recipient neurons. In a mouse model of stroke, treatment with extracellular mitochondria modified by O-GlcNAcylation reduces neuronal injury and improves neurologic deficits. In cerebrospinal fluid (CSF) samples from SAH patients, levels of O-GlcNAcylation in extracellular mitochondria correlate with better clinical outcomes. Conclusions These findings suggest that AGE-modification in extracellular mitochondria may induce danger signals, but O-GlcNAcylation can prevent glycation and improve the therapeutic efficacy of transplanted mitochondria in the CNS.
