Ischemic Boundary Zone Research Articles

NCAM mimetic peptide P2 synergizes with bone marrow mesenchymal stem cells in promoting functional recovery after stroke

The neural cell adhesion molecule (NCAM) promotes neural development and regeneration. Whether NCAM mimetic peptides could synergize with bone marrow mesenchymal stem cells (BMSCs) in stroke treatment deserves investigation. We found that the NCAM mimetic peptide P2 promoted BMSC proliferation, migration, and neurotrophic factor expression, protected neurons from oxygen-glucose deprivation through ERK and PI3K/AKT activation and anti-apoptotic mechanisms in vitro. Following middle cerebral artery occlusion (MCAO) in rats, P2 alone or in combination with BMSCs inhibited neuronal apoptosis and induced the phosphorylation of ERK and AKT. P2 combined with BMSCs enhanced neurotrophic factor expression and BMSC proliferation in the ischemic boundary zone. Moreover, combined P2 and BMSC therapy induced translocation of nuclear factor erythroid 2-related factor, upregulated heme oxygenase-1 expression, reduced infarct volume, and increased functional recovery as compared to monotreatments. Treatment with LY294002 (PI3K inhibitor) and PD98059 (ERK inhibitor) decreased the neuroprotective effects of combined P2 and BMSC therapy in MCAO rats. Collectively, P2 is neuroprotective while P2 and BMSCs work synergistically to improve functional outcomes after ischemic stroke, which may be attributed to mechanisms involving enhanced BMSC proliferation and neurotrophic factor release, anti-apoptosis, and PI3K/AKT and ERK pathways activation.

Abstract TMP109: Labeling And Isolating Cell Specific Neuronal Mitochondria And Their Functional Analysis In Mice Post Stroke

Introduction: In vivo neuronal mitochondria dysfunction post stroke has not been well investigated. Methods: Using Mito-tag mice in combine with local infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by neuronal promoters, we generated reporter mice expressing a hemagglutinin (HA)-tagged EGFP that localizes to mitochondrial outer membranes in synaptophysin (SYN) and calmodulin-kinase II (CaMKII) glutamatergic neurons localized to cerebral cortex. These mice were subjected to permanent middle cerebral artery occlusion and sacrificed 14 days post stroke. Fresh brain tissues were excised from the ischemic core (IC), ischemic boundary zone (IBZ) as well as from the homologous contralateral hemisphere, and the crude mitochondrial fractions (CMF) were extracted. Neuronal mitochondria were then isolated from the CMF by anti-HA magnetic beads (Thermo Scientific) and their specificity and oxidative phosphorylation (OXPHOS) complexes were confirmed by means of Western-blot. Seahorse assay was employed to measure oxygen consumption rate (OCR) of the isolated mitochondria as an indicator of mitochondrial function. Results: The bead pulled components were co-precipitated with GFP and enriched with mitochondrial markers, e.g. voltage-dependent anion channel, cytochrome C, and COX IV, but lacked the Golgi protein RCAS1 as well as endoplasmic reticulum markers: Heme-oxygenase 1 and Calnexin, indicating that specific neuronal mitochondria have been selectively isolated. Western-blot data showed that OXPHOS components in SYN + and CAMKII + neuronal mitochondria were significantly downregulated in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function measured by OCR (p<0.05, respectively, for both neuron types). These data suggesting dysfunction of neuronal mitochondria post stroke. Conclusions: We, for the first time, measure mitochondrial function in 2 types of neurons post stroke. By introducing specific neuronal promoter controlled Cre recombinase to Mito-tag mouse, mitochondria from individual neuronal linage cells can be efficiently labeled and isolated to investigate functional change post stroke.

Synergistic therapeutic effects of intracerebral transplantation of human modified bone marrow-derived stromal cells (SB623) and voluntary exercise with running wheel in a rat model of ischemic stroke

BackgroundMesenchymal stromal cell (MSC) transplantation therapy is a promising therapy for stroke patients. In parallel, rehabilitation with physical exercise could ameliorate stroke-induced neurological impairment. In this study, we aimed to clarify whether combination therapy of intracerebral transplantation of human modified bone marrow-derived MSCs, SB623 cells, and voluntary exercise with running wheel (RW) could exert synergistic therapeutic effects on a rat model of ischemic stroke.MethodsWistar rats received right transient middle cerebral artery occlusion (MCAO). Voluntary exercise (Ex) groups were trained in a cage with RW from day 7 before MCAO. SB623 cells (4.0 × 105 cells/5 μl) were stereotactically injected into the right striatum at day 1 after MCAO. Behavioral tests were performed at day 1, 7, and 14 after MCAO using the modified Neurological Severity Score (mNSS) and cylinder test. Rats were euthanized at day 15 after MCAO for mRNA level evaluation of ischemic infarct area, endogenous neurogenesis, angiogenesis, and expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). The rats were randomly assigned to one of the four groups: vehicle, Ex, SB623, and SB623 + Ex groups.ResultsSB623 + Ex group achieved significant neurological recovery in mNSS compared to the vehicle group (p < 0.05). The cerebral infarct area of SB623 + Ex group was significantly decreased compared to those in all other groups (p < 0.05). The number of BrdU/Doublecortin (Dcx) double-positive cells in the subventricular zone (SVZ) and the dentate gyrus (DG), the laminin-positive area in the ischemic boundary zone (IBZ), and the mRNA level of BDNF and VEGF in SB623 + Ex group were significantly increased compared to those in all other groups (p < 0.05).ConclusionsThis study suggests that combination therapy of intracerebral transplantation SB623 cells and voluntary exercise with RW achieves robust neurological recovery and synergistically promotes endogenous neurogenesis and angiogenesis after cerebral ischemia, possibly through a mechanism involving the up-regulation of BDNF and VEGF.

Antagonism of histamine H3 receptor promotes angiogenesis following focal cerebral ischemia.

Our previous study showed that H3 receptor antagonists reduced neuronal apoptosis and cerebral infarction in the acute stage after cerebral ischemia, but through an action independent of activation of histaminergic neurons. Because enhanced angiogenesis facilitates neurogenesis and neurological recovery after ischemic stroke, we herein investigated whether antagonism of H3R promoted angiogenesis after brain ischemia. Photothrombotic stroke was induced in mice. We showed that administration of H3R antagonist thioperamide (THIO, 10 mg·kg-1·d-1, i.p., from D1 after cerebral ischemia) significantly improved angiogenesis assessed on D14, and attenuated neurological defects on D28 after cerebral ischemia. Compared with wild-type mice, Hrh3-/- mice displayed more blood vessels in the ischemic boundary zone on D14, and THIO administration did not promote angiogenesis in these knockout mice. THIO-promoted angiogenesis in mice was reversed by i.c.v. injection of H3R agonist immepip, but not by H1 and H2 receptor antagonists, histidine decarboxylase inhibitor α-fluoromethylhistidine, or histidine decarboxylase gene knockout (HDC-/-), suggesting that THIO-promoted angiogenesis was independent of activation of histaminergic neurons. In vascular endothelial cells (bEnd.3), THIO (10-9-10-7 M) dose-dependently facilitated cell migration and tube formation after oxygen glucose deprivation (OGD), and H3R knockdown caused similar effects. We further revealed that H3R antagonism reduced the interaction between H3R and Annexin A2, while knockdown of Annexin A2 abrogated THIO-promoted angiogenesis in bEnd.3 cells after OGD. Annexin A2-overexpressing mice displayed more blood vessels in the ischemic boundary zone, which was reversed by i.c.v. injection of immepip. In conclusion, this study demonstrates that H3R antagonism promotes angiogenesis after cerebral ischemia, which is independent of activation of histaminergic neurons, but related to the H3R on vascular endothelial cells and its interaction with Annexin A2. Thus, H3R antagonists might be promising drug candidates to improve angiogenesis and neurological recovery after ischemic stroke.

Post-stroke treatment with argon preserved neurons and attenuated microglia/macrophage activation long-termly in a rat model of transient middle cerebral artery occlusion (tMCAO)

In a previous study from our group, argon has shown to significantly attenuate brain injury, reduce brain inflammation and enhance M2 microglia/macrophage polarization until 7 days after ischemic stroke. However, the long-term effects of argon have not been reported thus far. In the present study, we analyzed the underlying neuroprotective effects and potential mechanisms of argon, up to 30 days after ischemic stroke. Argon administration with a 3 h delay after stroke onset and 1 h after reperfusion demonstrated long-term neuroprotective effect by preserving the neurons at the ischemic boundary zone 30 days after stroke. Furthermore, the excessive microglia/macrophage activation in rat brain was reduced by argon treatment 30 days after ischemic insult. However, long-lasting neurological improvement was not detectable. More sensorimotor functional measures, age- and disease-related models, as well as further histological and molecular biological analyses will be needed to extend the understanding of argon’s neuroprotective effects and mechanism of action after ischemic stroke.

Zeb2/Axin2-Enriched BMSC-Derived Exosomes Promote Post-Stroke Functional Recovery by Enhancing Neurogenesis and Neural Plasticity.

Exosomes harvested from bone marrow-derived mesenchymal stromal cells (BMSCs) have shown treatment potential in many diseases. In vitro, Zeb2/Axin2 stimulated endogenous neurogenesis, which induced functional recovery after stroke. Here, we investigated whether the Zeb2/Axin2-enriched exosomes harvested from BMSCs transfected with a Zeb2/Axin2 overexpression plasmid would enhance neurological recovery. Compared with the control, both exosome treatments significantly improved functional recovery, and Zeb2/Axin2-enriched exosomes had significantly more improved effects on neurological function, neurogenesis, and neurite remodeling/neuronal dendrite plasticity than the control BMSC exosome treatment in a middle cerebral artery occlusion MCAO rat model. After stimulation with Zeb2/Axin2-enriched BMSC exosomes, the spatial memory and nerve function of MCAO rats showed marked recovery. The number of neurons was increased in the subventricularzone (SVZ), hippocampus, and cortex area, while the expression of nerve growth factors (NGF, BDNF, etc.) was upregulated. In the ischemic boundary zone, Zeb2/Axin2-enriched exosomes promoted synaptic remodeling by increasing the number of synapses and reversed the axonal loss of phosphorylated neurofilament (SMI-31) and synaptophysin (SYN) caused by ischemic injury, thus alleviating axonal demise and promoting synaptic proliferation. In vitro, Zeb2/Axin2-enriched exosomes significantly increased neurite branching and elongation of cultured cortical embryonic rat neurons under oxygen- and glucose-deprived (OGD) conditions. Moreover, Ex-Zeb2/Axin2-enriched exosomes downregulated the protein level of SOX10, endothelin-3/EDNRB, and Wnt/β-catenin expression. In conclusion, exosomes harvested from Ex-Zeb2/Axin2 BMSC could improve post-stroke neuroplasticity and functional recovery in MCAO rats by promoting proliferation and differentiation of neural stem cells. The mechanism may be related to the SOX10, Wnt/β-catenin, and endothelin-3/EDNRB pathways.

Differential Effects of Physical and Social Enriched Environment on Angiogenesis in Male Rats After Cerebral Ischemia/Reperfusion Injury.

Different housing conditions, including housing space and the physiological and social environment, may affect rodent behavior. Here, we examined the effects of different housing conditions on post-stroke angiogenesis and functional recovery to clarify the ambiguity about environmental enrichment and its components. Male rats in the model groups underwent right middle cerebral artery occlusion (MCAO) followed by reperfusion. The MCAO rats were divided into four groups: the physical enrichment (PE) group, the social enrichment (SE) group, the combined physical and social enrichment (PSE) group and the ischemia/reperfusion + standard conditioning (IS) group. The rats in the sham surgery (SS) group were housed under standard conditions. In a set of behavioral tests, including the modified Neurological Severity Score (mNSS), rotarod test, and adhesive removal test, we demonstrated that the animals in the enriched condition groups exhibited significantly improved neurological functions compared to those in the standard housing group. Smaller infarction volumes were observed in the animals of the PSE group by MRI detection. The enriched conditions increased the microvessel density (MVD) in the ischemic boundary zone, as revealed by CD31 immunofluorescent staining. The immunochemical and q-PCR results further showed that environmental enrichment increased the expression levels of angiogenic factors after ischemia/reperfusion injury. Our data suggest that all three enrichment conditions promoted enhanced angiogenesis and functional recovery after ischemia/reperfusion injury compared to the standard housing, while only exposure to the combination of both physical and social enrichment yielded optimal benefits.

Extracellular Vesicles Derived from Human Umbilical Cord Perivascular Cells Improve Functional Recovery in Brain Ischemic Rat via the Inhibition of Apoptosis

Background:Ischemic stroke, as a health problem caused by the reduced blood supply to the brain, can lead to the neuronal death. The number of reliable therapies for stroke is limited. MSCs exhibit therapeutic achievement. A major limitation of MSC application in cell therapy is the short survival span. MSCs affect target tissues through the secretion of many paracrine agents including EVs. This study aimed to investigate the effect of HUCPVCs-derived EVs on apoptosis, functional recovery, and neuroprotection. Methods:Ischemia was induced by MCAO in male Wistar rats. Animals were classified into sham, MCAO, MCAO + HUCPVC, and MCAO + EV groups. Treatments began at two hours after ischemia. Expressions of apoptotic-related proteins (BAX/BCl-2 and caspase-3 and -9), the amount of TUNEL-positive cells, neuronal density (MAP2), and dead neurons (Nissl staining) were assessed on day seven post MCAO. Results:Administration of EVs improved the sensorimotor function (p < 0.001) and reduced the apoptotic rate of Bax/Bcl-2 ratio (p < 0.001), as well as caspases and TUNEL-positive cells (p < 0.001) in comparison to the MCAO group. EV treatment also reduced the number of dead neurons and increased the number of MAP2+ cells in the IBZ (p < 0.001), as compared to the MCAO group. Conclusion: Our findings showed that HUCPVCs-derived EVs are more effective than their mother’s cells in improving neural function, possibly via the regulation of apoptosis in the ischemic rats. The strategy of cell-free extracts is, thus, helpful in removing the predicaments surrounding cell therapy in targeting brain diseases.

LincRNA-EPS in biomimetic vesicles targeting cerebral infarction promotes inflammatory resolution and neurogenesis

BackgroundInflammatory damage following stroke aggravates brain damage, resulting in long-term neurological sequelae. The purpose of this study was to identify ways to reduce inflammatory reactions and to accelerate neuron regeneration after cerebral apoplexy.MethodsWe formulated a biomimetic vesicle, the leukosome, constituted by liposome, artificial long intergenic noncoding RNA (lincRNA)-EPS, and membrane proteins derived from macrophages and their physical–chemical characteristics were evaluated. Migration distance and cytotoxic levels were measured to determine the effect of lncEPS-leukosomes on lipopolysaccharide-activated microglia. An in vivo transient middle cerebral artery occlusion/reperfusion (tMCAO) model was established in mice, which were treated with lncEPS-leukosomes. Vesicle seepage, infiltration of inflammatory cells, cytotoxic levels in the cerebrospinal fluid, and neural stem cell (NSC) density were measured.ResultsBiomimetic vesicles with a homogeneous size increased lincRNA-EPS levels in activated microglia by 77.9%. In vitro studies showed that lincRNA-EPS inhibited the migration and cytotoxic levels of activated microglia by 63.2% and 43.6%, respectively, which promoted NSC proliferation and anti-apoptotic ability. In vivo data showed that leukosomes targeted to inflamed sites and lncEPS-leukosomes decreased the infiltration of inflammatory cells and cytotoxic levels by 81.3% and 48.7%, respectively. In addition, lncEPS-leukosomes improved neuron density in the ischemic core and boundary zone after tMCAO.ConclusionsThe biomimetic vesicles formulated in this study targeted inflammatory cells and accelerated neuron regeneration by promoting inflammation resolution. This study may provide a promising treatment approach for accelerated neuron regeneration after cerebral apoplexy.

Neuroprotective effects of saffron on the late cerebral ischemia injury through inhibiting astrogliosis and glial scar formation in rats

This study is to explore the neuroprotective effects and involved glial scar of saffron (Crocus sativus L.) on the late cerebral ischemia in rats. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in Sprague Dawley rats that were randomly divided into sham group, MCAO group, edaravone group (as a positive control) and saffron groups (saffron extract 30, 100, 300 mg/kg). Saffron was administered orally at 2 h at the first day and once daily from day 2 to 42 after ischemia. Behavioral changes were detected from day 43 to 46 after ischemia to evaluate the effects of saffron. Infarct volume, survival neuron density, activated astrocyte, and the thickness of glial scar were also detected. GFAP, neurocan, phosphocan, neurofilament expressions and inflammatory cytokine contents were detected by Western-blotting and ELISA methods, respectively. Saffron improved the body weight loss, neurological deficit and spontaneous activity. It also ameliorated anxiety-like state and cognitive dysfunction, which were detected by elevated plus maze (EPM), marble burying test (MBT) and novel object recognition test (NORT). Toluidine blue staining found that saffron treatment decreased the infarct volume and increased the neuron density in cortex in the ischemic boundary zone. The activated astrocyte number and the thickness of glial scar in the penumbra zone reduced after saffron treatment. Additionally, saffron decreased the contents of IL-6 and IL-1β, increased the content of IL-10 in the ischemic boundary zone. GFAP, neurocan, and phosphocan expressions in ischemic boundary zone and ischemic core zone all decreased after saffron treatment. Saffron exerted neuroprotective effects on late cerebral ischemia, associating with attenuating astrogliosis and glial scar formation after ischemic injury.

Improvement of Neurological Function in Rats with Ischemic Stroke by Adipose-derived Pericytes

Pericytes possess high multipotent features and cell plasticity, and produce angiogenic and neurotrophic factors that indicate their high regenerative potential. The aim of this study was to investigate whether transplantation of adipose-derived pericytes can improve functional recovery and neurovascular plasticity after ischemic stroke in rats. Rat adipose-derived pericytes were isolated from subcutaneous adipose tissue by fluorescence-activated cell sorting. Adult male Wistar rats were subjected to 90 min of middle cerebral artery occlusion followed by intravenous injection of rat adipose-derived pericytes 24 h later. Functional recovery evaluations were performed at 1, 7, 14, and 28 days after injection of rat adipose-derived pericytes. Angiogenesis and neurogenesis were examined in rat brains using immunohistochemistry. It was observed that intravenous injection of adipose-derived pericytes significantly improved recovery of neurological function in rats with stroke compared to phosphate-buffered saline–treated controls. Immunohistochemical analysis revealed that the number of blood capillaries was significantly increased along the ischemic boundary zone of the cortex and striatum in stroke rats treated with adipose-derived pericytes. In addition, treatment with adipose-derived pericytes increased the number of doublecortin positive neuroblasts. Our data suggest that transplantation of adipose-derived pericytes can significantly improve the neurologic status and contribute to neurovascular remodeling in rats after ischemic stroke. These data provide a new insight for future cell therapies that aim to treat ischemic stroke patients.

Effects of neural stem cell-derived extracellular vesicles on neuronal protection and functional recovery in the rat model of middle cerebral artery occlusion.

Stroke imposes a long-term neurological disability with limited effective treatments available for neuronal recovery. Transplantation of neural stem cells (NSCs) is reported to improve functional outcomes in the animal models of brain ischemia. However, the use of cell therapy is accompanied by adverse effects, so research is growing to use cell-free extracts such as extracellular vesicles (EVs) for targeting brain diseases. In the current study, male Wistar albino rats (20 months old) were subjected to middle cerebral artery occlusion (MCAO). Then, EVs (30 μg) were injected at 2 hours after stroke onset via an intracerebroventricular (ICV) route. Measurements were done at day 7 post-MCAO. EVs administration reduced lesion volume and steadily improved spontaneous locomotor activity. EVs administration also reduced microgliosis (ionized calcium-binding adaptor molecule 1 (Iba1)+ cells) and apoptotic (terminal-deoxynucleotidyl transferase mediated nick end labelling [TUNEL]) positive cells and increased neuronal survival (neuronal nuclear (NeuN)+ cells) in the ischemic boundary zone (IBZ). However, it had no effect on neurogenesis within the sub-ventricular zone (SVZ) but decreased cellular migration toward the IBZ (doublecortin (DCX)+ cells). The results of this study showed neuroprotective and restorative mechanisms of NSC-EVs administration, which may offer new avenues for therapeutic intervention of brain ischemia. SIGNIFICANCE OF THE STUDY: Based on our results, EVs administration can effectively reduce microglial density and neuronal apoptosis, thereby steadily improves functional recovery after MCAO. These findings provide the beneficial effect of NSC-EVs as a new biological treatment for stroke.

MiR-126 Mediates Brain Endothelial Cell Exosome Treatment-Induced Neurorestorative Effects After Stroke in Type 2 Diabetes Mellitus Mice.

Background and Purpose- Stroke patients with type 2 diabetes mellitus (T2DM) exhibit increased vascular and white matter damage and have worse prognosis compared with nondiabetic stroke patients. We investigated the neurorestorative effects of exosomes derived from mouse brain endothelial cells (EC-Exo) as treatment for stroke in T2DM mice and investigated the role of miR-126 in mediating EC-Exo-derived therapeutic benefits in T2DM-stroke mice. Methods- Adult, male BKS.Cg-m+/+Leprdb/J (T2DM) mice were subjected to photothrombotic stroke model. T2DM mice were intravenously injected at 3 days after stroke with (1) PBS; (2) liposome mimic (vehicle control, 3×1010); (3) EC-Exo (3×1010); (4) knockdown of miR-126 in EC-Exo (miR-126-/- EC-Exo, 3×1010). Behavioral and cognitive tests were performed, and mice were sacrificed at 28 days after stroke. Results- Compared with non-DM stroke mice, T2DM-stroke mice exhibit significantly decreased serum and brain tissue miR-126 expression. Endothelial cells and EC-Exo contain high levels of miR-126 compared with other cell types or exosomes derived from other types of cells, respectively (smooth muscle cells, astrocytes, and marrow stromal cells). Compared with PBS or liposome mimic treatment, EC-Exo treatment of T2DM-stroke mice significantly improves neurological and cognitive function, increases axon density, myelin density, vascular density, arterial diameter, as well as induces M2 macrophage polarization in the ischemic boundary zone. MiR-126-/- EC-Exo treatment significantly decreases miR-126 expression in serum and brain, as well as attentuates EC-Exo treatment-induced functional improvement and does not significantly increase axon and myelin density, vascular density, arterial diameter or induce M2 macrophage polarization in T2DM-stroke mice. In vitro, EC-Exo treatment significantly increases primary cortical neuron axonal outgrowth and increases endothelial capillary tube formation whereas miR-126-/- EC-Exo attentuates EC-Exo induced capillary tube formation and axonal outgrowth. Conclusions- EC-Exo treatment of stroke promotes neurorestorative effects in T2DM mice. MiR-126 may mediate EC-Exo-induced neurorestorative effects in T2DM mice. Visual Overview- An online visual overview is available for this article.

Post-stroke treatment with argon attenuated brain injury, reduced brain inflammation and enhanced M2 microglia/macrophage polarization: a randomized controlled animal study

BackgroundIn recent years, argon has been shown to exert neuroprotective effects in an array of models. However, the mechanisms by which argon exerts its neuroprotective characteristics remain unclear. Accumulating evidence imply that argon may exert neuroprotective effects via modulating the activation and polarization of microglia/macrophages after ischemic stroke. In the present study, we analyzed the underlying neuroprotective effects of delayed argon application until 7 days after reperfusion and explored the potential mechanisms.MethodsTwenty-one male Wistar rats underwent transient middle cerebral artery occlusion or sham surgery randomly for 2 h using the endoluminal thread model. Three hours after transient middle cerebral artery occlusion induction and 1 h after reperfusion, animals received either 50% vol Argon/50% vol O2 or 50% vol N2/50% vol O2 for 1 h. The primary outcome was the 6-point neuroscore from 24 h to d7 after reperfusion. Histological analyses including infarct volume, survival of neurons (NeuN) at the ischemic boundary zone, white matter integrity (Luxol Fast Blue), microglia/macrophage activation (Iba1), and polarization (Iba1/Arginase1 double staining) on d7 were conducted as well. Sample size calculation was performed using nQuery Advisor + nTerim 4.0. Independent t test, one-way ANOVA and repeated measures ANOVA were performed, respectively, for statistical analysis (SPSS 23.0).ResultsThe 6-point neuroscore from 24 h to d7 after reperfusion showed that tMCAO Ar group displayed significantly improved neurological performance compared to tMCAO N2 group (p = 0.026). The relative numbers of NeuN-positive cells in the ROIs of tMCAO Ar group significantly increased compared to tMCAO N2 group (p = 0.010 for cortex and p = 0.011 for subcortex). Argon significantly suppressed the microglia/macrophage activation as revealed by Iba1 staining (p = 0.0076) and promoted the M2 microglia/macrophage polarization as revealed by Iba1/Arginase 1 double staining (p = 0.000095).ConclusionsArgon administration with a 3 h delay after stroke onset and 1 h after reperfusion significantly alleviated neurological deficit within the first week and preserved the neurons at the ischemic boundary zone 7 days after stroke. Moreover, argon reduced the excessive microglia/macrophage activation and promoted the switch of microglia/macrophage polarization towards the anti-inflammatory M2 phenotype. Studies making efforts to further elucidate the protective mechanisms and to benefit the translational application are of great value.

Constraint-induced movement therapy enhances angiogenesis and neurogenesis after cerebral ischemia/reperfusion.

Constraint-induced movement therapy after cerebral ischemia stimulates axonal growth by decreasing expression levels of Nogo-A, RhoA, and Rho-associated kinase (ROCK) in the ischemic boundary zone. However, it remains unclear if there are any associations between the Nogo-A/RhoA/ROCK pathway and angiogenesis in adult rat brains in pathological processes such as ischemic stroke. In addition, it has not yet been reported whether constraint-induced movement therapy can promote angiogenesis in stroke in adult rats by overcoming Nogo-A/RhoA/ROCK signaling. Here, a stroke model was established by middle cerebral artery occlusion and reperfusion. Seven days after stroke, the following treatments were initiated and continued for 3 weeks: forced limb use in constraint-induced movement therapy rats (constraint-induced movement therapy group), intraperitoneal infusion of fasudil (a ROCK inhibitor) in fasudil rats (fasudil group), or lateral ventricular injection of NEP1-40 (a specific antagonist of the Nogo-66 receptor) in NEP1-40 rats (NEP1-40 group). Immunohistochemistry and western blot assay results showed that, at 2 weeks after middle cerebral artery occlusion, expression levels of RhoA and ROCK were lower in the ischemic boundary zone in rats treated with NEP1-40 compared with rats treated with ischemia/reperfusion or constraint-induced movement therapy alone. However, at 4 weeks after middle cerebral artery occlusion, expression levels of RhoA and ROCK in the ischemic boundary zone were markedly decreased in the NEP1-40 and constraint-induced movement therapy groups, but there was no difference between these two groups. Compared with the ischemia/reperfusion group, modified neurological severity scores and foot fault scores were lower and time taken to locate the platform was shorter in the constraint-induced movement therapy and fasudil groups at 4 weeks after middle cerebral artery occlusion, especially in the constraint-induced movement therapy group. Immunofluorescent staining demonstrated that fasudil promoted an immune response of nerve-regeneration-related markers (BrdU in combination with CD31 (platelet endothelial cell adhesion molecule), Nestin, doublecortin, NeuN, and glial fibrillary acidic protein) in the subventricular zone and ischemic boundary zone ipsilateral to the infarct. After 3 weeks of constraint-induced movement therapy, the number of regenerated nerve cells was noticeably increased, and was accompanied by an increased immune response of tight junctions (claudin-5), a pericyte marker (α-smooth muscle actin), and vascular endothelial growth factor receptor 2. Taken together, the results demonstrate that, compared with fasudil, constraint-induced movement therapy led to stronger angiogenesis and nerve regeneration ability and better nerve functional recovery at 4 weeks after cerebral ischemia/reperfusion. In addition, constraint-induced movement therapy has the same degree of inhibition of RhoA and ROCK as NEP1-40. Therefore, constraint-induced movement therapy promotes angiogenesis and neurogenesis after cerebral ischemia/reperfusion injury, at least in part by overcoming the Nogo-A/RhoA/ROCK signaling pathway. All protocols were approved by the Institutional Animal Care and Use Committee of China Medical University, China on December 9, 2015 (approval No. 2015PS326K).

MiRNA-27b Regulates Angiogenesis by Targeting AMPK in Mouse Ischemic Stroke Model

Stroke is a leading cause of mortality and serious disability worldwide with limited treatment options. Angiogenesis has been reported to be involved in post-stroke recovery. Although the molecular mechanisms that regulate angiogenesis remain ambiguous, microRNAs have emerged as effective regulators of angiogenesis, involved in neurological function outcome. The present study aims to investigate the regulatory effects of miRNA-27b on post-stroke angiogenesis. In primary cultured brain microvascular endothelial cells (BMECs), the inhibition of miRNA-27b induced the activation of adenosine monophosphate-activated protein kinase (AMPK), which increased tube formation and migration. This action was attenuated when AMPKα2 was knocked down. Mice were subjected to middle cerebral artery occlusion (MCAo) surgery and administrated with Lentivirus miR-27b inhibitor. Enhanced angiogenesis in ischemic boundary zone (IBZ) was observed, and the neurological outcome during the entire study period was improved. The number of phosphate-AMPKα2+ cells that co-expressed endothelial cell marker CD31 was significantly increased. Taken together, the present study demonstrated that downregulated miRNA-27b promoted recovery after ischemic stroke via AMPK stimulus.

Combination therapy of human bone marrow\u2013derived mesenchymal stem cells and minocycline improves neuronal function in a rat middle cerebral artery occlusion model

BackgroundThe positive effects of human bone marrow–derived mesenchymal stem cells (hBM-MSCs) and minocycline on ischemic stroke models have been well described through numerous studies. The aim of this study was to evaluate the effectiveness of combination therapy of hBM-MSCs with minocycline in a middle cerebral artery occlusion rat model.MethodsForty male Sprague-Dawley rats were enrolled in this study. After right middle cerebral artery occlusion, rats were randomly assigned to one of four groups: control, minocycline, hBM-MSCs, or hBM-MSCs with minocycline. Rotarod test, adhesive-removal test, and modified neurological severity score grading were performed before and 1, 7, 14, 21, and 28 days after right middle cerebral artery occlusion. All rats were sacrificed at day 28. The volume of the infarcted area was measured with triphenyl tetrazolium chloride staining. Neuronal nuclear antigen (NeuN)- and vascular endothelial growth factor (VEGF)-positive cells in the ischemic boundary zone were assessed by immunofluorescence.ResultsNeurological outcome in the adhesive-removal test and rotarod test and modified neurological severity score were better in the combination therapy group than in the monotherapy and control groups. The volume of the infarcted area was smaller in the combination group compared with the others. The proportions of NeuN- and VEGF-positive cells in the ischemic boundary were highest in the combination therapy group.ConclusionsEarly combination therapy of hBM-MSCs with minocycline in an ischemic stroke model may enhance neurological recovery, reduce the volume of the infarcted area, and promote the expression of NeuN and VEGF in ischemic boundary cells.

Survivin overexpression via adeno-associated virus vector Rh10 ameliorates ischemic damage after middle cerebral artery occlusion in rats.

Survivin, a member of the inhibitors of apoptosis protein gene family, inhibits the activity of caspase, leading to a halt of the apoptotic process. Our study focused on the neuroprotective effect of survivin after transient middle cerebral artery occlusion (MCAO) with intraparenchymal administration of an adeno-associated virus (AAV) vector. His-tagged survivin was cloned and packaged into the AAV-rh10 vector. Four-week-old Sprague-Dawley rats were injected with 4×109 vg of AAV-GFP or AAV-His-survivin into the right striatum and were treated 3weeks later with transient MCAO for 90min. Twenty-four hours after MCAO, functional and histological analyses of the rats were performed. The result showed that rats that had been treated with AAV-green fluorescent protein (GFP) and those that had been treated with AAV-His-survivin did not show a significant difference in neurological scores 24hr after MCAO, however, infarction volume was significantly reduced in the AAV-His-survivin group compared to that in the AAV-GFP group. Although the neutrophil marker myeloperoxidase did not show a significant difference in the ischemic boundary zone, cells positive for active caspase-3 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling were significantly decreased in the AAV-His-survivin group. In conclusion, survivin overexpression in the ischemic boundary zone induced by using an AAV vector has the potential for amelioration of ischemic damage via an antiapoptotic mechanism.

Treadmill exercise promotes the expression of TGF-β1 and Smad3 protein, inhibiting cell apoptosis after cerebral ischemia

Objective To observe the effect of treadmill exercise on the expression of transforming growth factor-β1 (TGF-β1) and its receptor Smad3 protein as well as on cell apoptosis in the ischemic boundary zone, so as to explore how exercise promotes the recovery of neurological function after cerebral ischemia. Methods Thirty adult male Sprague-Dawley rats were randomly divided into a sham group (n=6), a model group (n=12) and an exercise group (n=12). A modified Longa′s method was used to establish an animal model of cerebral ischemia by occluding the right middle cerebral artery in the rats of the model and exercise groups. Those of the sham group were subjected to the same surgical procedure except that no thread was inserted. After 24h the exercise group began treadmill training, while the other two groups were left on the treadmill without training. Modified neurological severity scores (mNSSs) were used to quantify the rats′ neurological functioning on the 3rd, 7th and 14th day after the surgery. The ischemic boundary zone tissue was then dissected to detect the expression of TGF-β1 and Smad3 protein using western blotting. Cell apoptosis was detected by TUNEL staining. Results The average mNSS scores of the exercise group on the 7th and the 14th day were significantly lower than those of the model group at the same time points. The average expression level of TGF-β1 and Smad3 protein in the exercise group was significantly higher than in the model group. The number of TUNEL-positive cells in the exercise group was significantly lower than in the model group on the 14th day. Conclusions Treadmill exercise can improve the recovery of neurological function after cerebral ischemia. It may be partly due to upregulating the expression of TGF-β1 and Smad3 protein, which inhibit cell apoptosis in the ischemic boundary zone. Key words: Cerebral ischemia; Exercise; Transforming growth factor; Smad3 protein; Apoptosis

AMPA Receptor Antagonist Perampanel Ameliorates Post-Stroke Functional and Cognitive Impairments

Perampanel (PER), a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor antagonist, clinically used for seizure control, has been reported to exert neuroprotective effects in experimental models of neurodegenerative diseases. However, few studies have investigated the therapeutic effects of PER in brain injury including stroke. Our aim was to investigate the neuroprotective potential of PER using a rat transient middle cerebral artery occlusion (MCAO) model. Sprague–Dawley rats underwent 90-min MCAO followed by intraperitoneal PER administration at a dose of 1.5 mg/kg. Infarct volumes, neurological deficits, and immunological analyses were performed at 7 days after MCAO. PER significantly reduced infarct volumes (p < 0.05) and improved motor function (p < 0.05) compared with vehicle. Immunological analysis showed that PER significantly inhibited microglial activation, pro-inflammatory cytokine expression, and oxidative stress compared with vehicle. Moreover, PER suppressed neurodegeneration in the cortical ischemic boundary zone, via downregulation of Bcl-2-associated x and upregulation of Bcl-extra-large with Akt activation. In addition, post-stroke secondary neuronal damage and cognitive impairments, using the Y-maze test, were assessed 30 days after MCAO. PER significantly improved spatial working memory, which was accompanied by hippocampal CA1 neuronal loss and cortical thinning, compared with vehicle. These results indicate that PER attenuates infarct volumes and motor function deficits possibly through its anti-inflammatory, antioxidant, and anti-apoptotic activities, mediated via activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathways in the acute ischemic phase, and further ameliorates post-stroke cognitive impairments via the suppression of secondary neuronal damage in the chronic ischemic phase.