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Rationale: Microglia play a critical role in modulating cell death and neurobehavioral recovery in response to brain injury either by direct cell-cell interaction or indirect secretion of trophic factors. Exosomes secreted from cells are well documented to deliver bioactive molecules to recipient cells to modulate cell function. Here, we aimed to identify whether M2 microglia exert neuroprotection after ischemic attack through an exosome-mediated cell-cell interaction. Methods: M2 microglia-derived exosomes were intravenously injected into the mouse brain immediately after middle cerebral artery occlusion. Infarct volume, neurological score, and neuronal apoptosis were examined 3 days after ischemic attack. Exosome RNA and target protein expression levels in neurons and brain tissue were determined for the mechanistic study. Results: Our results showed that the M2 microglia-derived exosomes were taken up by neurons in vitro and in vivo . M2 microglia-derived exosome treatment attenuated neuronal apoptosis after oxygen-glucose deprivation (p<0.05). In vivo results showed that M2 microglia-derived exosome treatment significantly reduced infarct volume and attenuated behavioral deficits 3 days after transient brain ischemia (p<0.05), whereas injection of miR-124 knockdown (miR-124k/d) M2 microglia-derived exosomes partly reversed the neuroprotective effect. Our mechanistic study further demonstrated that ubiquitin-specific protease 14 (USP14) was the direct downstream target of miR-124. Injection of miR-124k/d M2 exosomes plus the USP14 inhibitor, IU1, achieved comparable neuroprotective effect as injection of M2 exosomes alone. Conclusions: We demonstrated that M2 microglia-derived exosomes attenuated ischemic brain injury and promoted neuronal survival via exosomal miR-124 and its downstream target USP14. M2 microglia-derived exosomes represent a promising avenue for treating ischemic stroke.

Background and Purpose-Inflammatory response plays a critical role in propagating tissue damage after focal cerebral ischemia. CXCL12 is a key chemokine for leukocyte recruitment. However, the role of CXCL12 and its receptor CXCR4 in ischemia-induced inflammatory response is unclear. Here we use the pharmacological antagonist of CXCR4, AMD3100, to investigate the function of CXCL12/CXCR4 in regulating inflammatory response during acute ischemia. Methods-Adult male CD-1 mice (n=184) underwent permanent suture middle cerebral artery occlusion (MCAO). AMD3100 was injected for 3 days (1 mg/kg/day) after MCAO. Brain water content, infarct volume, neurological score, and myeloperoxidase (MPO) expression and activity were examined at 24, 48, and 72 hours after MCAO. Proinflammatory cytokine RNA and protein levels in brain tissue were measured by RT-PCR and enzyme linked immunosorbent assay. Results-Neurological score was greatly improved in AMD3100-treated mice compared with the control mice 3 days after MCAO (P<0.05). Brain edema-induced change of water content, IgG protein leakage, Evans blue extravasation, occludin, and ZO-1 expression in ipsilateral hemisphere were alleviated by acute treatment of AMD3100. MPO expression and activity revealed that AMD3100 profoundly reduced the number of MPO-positive cells in the ischemic region (P<0.05). It also attenuated proinflammatory cytokines including interleukin 6, tumor necrosis factor α, and interferon γ; their mRNA and protein levels changed accordingly compared with the controls (P<0.05). Conclusions-CXCR4 antagonist AMD3100 significantly suppressed inflammatory response and reduced blood-brain barrier disruption after MCAO. AMD3100 attenuated ischemia-induced acute inflammation by suppressing leukocyte migration and infiltration, in addition to reducing proinflammatory cytokine expression in the ischemic region.

IMPORTANCE Some uncertainty about the clinical value and dosing of atropine for the treatment of myopia in children remains. OBJECTIVE To evaluate the efficacy vs the adverse effects of various doses of atropine in the therapy for myopia in children. DATA SOURCES Data were obtained from PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials, from inception to April 30, 2016. The reference lists of published reviews and clinicaltrials.gov were searched for additional relevant studies. Key search terms included myopia, refractive errors, and atropine. Only studies published in English were included. STUDY SELECTION Randomized clinical trials and cohort studies that enrolled patients younger than 18 years with myopia who received atropine in at least 1 treatment arm and that reported the annual rate of myopia progression and/or any adverse effects of atropine therapy were included in the analysis. DATA EXTRACTION AND SYNTHESIS Two reviewers independently abstracted the data. Heterogeneity was statistically quantified by Q, H, and I 2 statistics, and a meta-analysis was performed using the random-effects model. The Cochrane Collaboration 6 aspects of bias and the Newcastle-Ottawa Scale were used to assess the risk for bias. MAIN OUTCOMES AND MEASURES The primary outcome was a difference in efficacy and the presence of adverse effects at different doses of atropine vs control conditions. The secondary outcomes included the differences in adverse effects between Asian and white patients. RESULTS Nineteen unique studies involving 3137 unique children were included in the analysis. The weighted mean differences between the atropine and control groups in myopia progression were 0.50 diopters (D) per year (95% CI, 0.24-0.76 D per year) for low-dose atropine, 0.57 D per year (95% CI, 0.43-0.71 D per year) for moderate-dose atropine, and 0.62 D per year (95% CI, 0.45-0.79 D per year) for high-dose atropine (P < .001), which translated to a high effect size (Cohen d, 0.97, 1.76, and 1.94, respectively). All doses of atropine, therefore, were equally beneficial with respect to myopia progression (P = .15). High-dose atropine were associated with more adverse effects, such as the 43.1% incidence of photophobia compared with 6.3% for low-dose atropine and 17.8% for moderate-dose atropine (χ 2 2 = 7.05; P = .03). In addition, differences in the incidence of adverse effects between Asian and white patients were not identified (χ 2 1 = 0.81; P = .37 for photophobia). CONCLUSIONS AND RELEVANCE This meta-analysis suggests that the efficacy of atropine is dose independent within this range, whereas the adverse effects are dose dependent.

Recent studies have demonstrated that exosomal microRNAs (miRNAs) are novel biomarkers and therapeutic targets for various diseases including vascular disease. However, specific exosomal miRNAs expression in stroke patients has not been reported yet. Here, we explored whether circulating exosomal miRNAs can serve as potential biomarkers for the diagnosis of acute ischemic stroke and discussed the potential for clinical application. Blood samples were collected from acute ischemic stroke patients within the first 72 h (n = 50). Circulating exosomes were exacted by Exoquick exosome isolation kit and characterized by transmission electron microscopy. Western blot was performed to assess the expression of exosomal protein makers. Exosomal miRNA-223 (miR-223) was detected by RT-PCR assay. The relationship between the expression levels of miR-223 and National Institutes of Health Stroke Scale (NIHSS) scores, brain infarct volume, and neurological outcomes were analyzed. Circulating exosomes were isolated and the size of vesicles ranged between 30 and 100 nm. The identification of exosomes was further confirmed by the detection of specific exosomal protein markers CD9, CD63, and Tsg101. Exosomal miR-223 in acute ischemic stroke patients was significantly upregulated compared to control group (p < 0.001). Exosomal miR-223 level was positively correlated with NIHSS scores (r = 0.31, p = 0.03). Exosomal miR-223 expression in stroke patients with poor outcomes was higher than those with good outcomes (p < 0.05). Increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes. Future studies with large sample are needed to assess the clinical application of exosomal miR-223 as a novel biomarker for ischemic stroke diagnosis.

Rationale: Cerebral ischemia upregulates aquaporin-4 expression, increases blood-brain barrier (BBB) permeability, and induces brain edema. Mesenchymal stem cells (MSCs) can repress inflammatory cytokines and show great potential for ischemic stroke therapy. However, the effect of MSCs regarding the protection of ischemia-induced BBB break down is unknown. Objective: We test whether MSCs therapy protects BBB integrity and explore the molecular mechanisms of aquaporin-4 on BBB integrity. Methods and Results: Two hundred and twenty-eight adult CD1 male mice underwent 90 minutes transient middle cerebral artery occlusion and received 2 3 10 5 MSCs intracranial transplantation. The neurological severity score was improved and both ischemia-induced brain edema and BBB leakage were reduced in MSC-treated mice. MSCs therapy reduced astrocyte apoptosis and inhibited ischemia-induced aquaporin-4 upregulation. In addition, small-interfering RNA knockdown of aquaporin-4 after cerebral ischemia effectively reduced aquaporin-4 expression, brain edema, BBB leakage, and astrocyte apoptosis. Conditional medium from lipopolysaccharide (LPS)-activated microglia enhanced aquaporin-4 expression, p38 and JNK phosphorylation, and apoptosis of cultured astrocytes. MSC treatment reduced the expression of inflammatory cytokines in LPS-activated microglia, and subsequently reduced aquaporin-4 expression and apoptosis of astrocytes. Knockdown of aquaporin-4 in cultured astrocytes also reduced apoptosis. Treatment with p38 and JNK inhibitors showed that p38, but not the JNK signaling pathway, was responsible for the aquaporin-4 upregulation. Conclusion: MSCs protected BBB integrity by reducing the apoptosis of astrocytes after ischemic attack, which was due to the attenuation of inflammatory response and downregulation of aquaporin-4 expression via p38 signaling pathway. STEM CELLS 2014;32:3150-3162

BackgroundMetformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms.MethodsNinety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways.ResultsInfarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect.ConclusionsMetformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-014-0177-4) contains supplementary material, which is available to authorized users.

Mesenchymal stem cell (MSC) transplantation has been shown to be beneficial in treating cerebral ischemia. However, such benefit is limited by the low survival of transplanted MSCs in an ischemic microenvironment. Previous studies showed that melatonin pretreatment can increase MSC survival in the ischemic kidney. However, whether it will improve MSC survival in cerebral ischemia is unknown. Our study examined the effect of melatonin pretreatment on MSCs under ischemia-related conditions in vitro and after transplantation into ischemic rat brain. Results showed that melatonin pretreatment greatly increased survival of MSCs in vitro and reduced their apoptosis after transplantation into ischemic brain. Melatonin-treated MSCs (MT-MSCs) further reduced brain infarction and improved neurobehavioral outcomes. Angiogenesis, neurogenesis, and the expression of vascular endothelial growth factor (VEGF) were greatly increased in the MT-MSC-treated rats. Melatonin treatment increased the level of p-ERK1/2 in MSCs, which can be blocked by the melatonin receptor antagonist luzindole. ERK phosphorylation inhibitor U0126 completely reversed the protective effects of melatonin, suggesting that melatonin improves MSC survival and function through activating the ERK1/2 signaling pathway. Thus, stem cells pretreated by melatonin may represent a feasible approach for improving the beneficial effects of stem cell therapy for cerebral ischemia.