Background and Purpose Type-2 diabetes mellitus (T2DM) is a major comorbidity that exacerbates ischemic brain injury and worsens functional outcome after stroke. T2DM is known to aggravate white matter impairment but the underlying mechanism is not completely understood. This study was designed to test the hypothesis that T2DM impedes post-stroke white matter recovery by suppressing both oligodendrogenesis and beneficial microglia/macrophage responses. Methods Permanent distal middle cerebral artery occlusion was performed in wild-type, homozygous diabetic db/db, and heterozygous db/+ mice. The adhesive removal, open field, and Morris water maze tests were used to assess neurobehavioral outcomes. Neuronal tissue loss, white matter damage, oligodendrogenesis, and microglia/macrophage responses were evaluated up to 35d after stroke. The functional integrity of white matter was measured by electrophysiology. Primary microglia-oligodendrocyte co-cultures were used for additional mechanistic studies. Results T2DM exacerbated structural damage and impaired conduction of compound action potentials in white matter 35d after stroke. The deterioration in white matter integrity correlated with poor sensorimotor performance. Furthermore, T2DM impaired the proliferation of oligodendrocyte precursor cells (OPCs) and the generation of new myelinating oligodendrocytes. T2DM also promoted a shift of microglia/macrophage phenotype toward the pro-inflammatory modality. Co-culture studies confirmed that microglia/macrophage polarization toward the pro-inflammatory phenotype under high glucose conditions suppressed OPC differentiation. Conclusions Deterioration of white matter integrity and impairments in oligodendrogenesis after stroke are associated with poor long-term functional outcomes in experimental diabetes mellitus. High glucose concentrations may shift microglia/macrophage polarization toward a pro-inflammatory phenotype, significantly impairing OPC differentiation and white matter repair.

MicroRNA-181 (miR-181) is highly expressed in the brain, and downregulated in miRNA expression profiles of acute ischemic stroke patients. However, the roles of miR-181c in stroke are not known. The clinical relevance of miR-181c in acute stroke patients was evaluated by real-time PCR and correlation analyses. Proliferation and apoptosis of BV2 microglial cells and Neuro-2a cells cultured separately or together under oxidative stress or inflammation were assessed with the Cell Counting Kit-8 and by flow cytometry, respectively. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in C57/BL6 mice, and cerebral infarct volume, microglia activation, and expression of pro-apoptotic factors were evaluated by 2,3,5-triphenyl-2H-tetrazolium chloride staining, immunocytochemistry, and western blotting, respectively. Plasma levels of miR-181c were decreased in stroke patients relative to healthy individuals, and were positively correlated with neutrophil number and blood platelet count and negatively correlated with lymphocyte number. Lipopolysaccharide (LPS)/hydrogen peroxide (H2O2) treatment inhibited BV2 microglia proliferation without inducing apoptosis, while miR-181c reduced proliferation but increased the apoptosis of these cells with or without LPS/H2O2 treatment. LPS/H2O2 induced apoptosis in Neuro-2a cells co-cultured with BV2 cells, an effect that was potentiated by miR-181c. In the MCAO model, miR-181c agomir modestly increased infarct volume, markedly decreased microglia activation and B cell lymphoma-2 expression, and increased the levels of pro-apoptotic proteins in the ischemic brain. Our data indicate that miR-181c contributes to brain injury in acute ischemic stroke by promoting apoptosis of microglia and neurons via modulation of pro- and anti-apoptotic proteins.

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Background and Purpose T helper cell 1 (Th1)‐skewed neurotoxicity contributes to the poor outcome of stroke in rodents. Here, we have elucidated the mechanism of the Th1/Th2 shift in acute ischaemic stroke (AIS) patients at hyperacute phase and have looked for a miRNA‐based therapeutic target. Experimental Approach MiR‐494 levels in blood from AIS patients and controls were measured by real‐time PCR. C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and cortical neurons were subjected to oxygen–glucose deprivation. Luciferase reporter system, chromatin immunoprecipitation sequencing (ChIP‐Seq), and ChIP‐PCR were used to uncover possible mechanisms. Key Results In lymphocytes from AIS patients, there was a Th1/Th2 shift and histone deacetylase 2 (HDAC2) was markedly down‐regulated. ChIP‐seq showed that HDAC2 binding sites were enriched in regulation of Th1 cytokine production, and ChIP‐PCR confirmed that HDAC2 binding was changed at the intron of STAT4 and the promoter of T‐box transcription factor 21 (T‐bet) in lymphocytes from AIS patients. MiR‐494 was the most significantly increased miRNA in lymphocytes from AIS patients, and miR‐494‐3p directly targeted HDAC2. A strong association existed between miR‐494 and Th1 cytokines, and neurological deficit as measured by the National Institute of Health Stroke Scale (NIHSS) in AIS patients. In vitro and in vivo experiments showed that antagomir‐494 reduced Th1 shift‐mediated neuronal and sensorimotor functional damage in the mouse model of ischaemic stroke, via the HDAC2‐STAT4 pathway. Conclusion and Implications We demonstrated that miR‐494 inhibition prevented Th1‐skewed neurotoxicity through regulation of the HDAC2‐STAT4 cascade.

Emerging evidence suggests that tissue plasminogen activator (tPA), currently the only FDA-approved medication for ischemic stroke, exerts important biological actions on the CNS besides its well-known thrombolytic effect. In this study, we investigated the role of tPA on primary neurons in culture and on brain recovery and plasticity after ischemic stroke in mice. Treatment with recombinant tPA stimulated axonal growth in culture, an effect independent of its protease activity and achieved through epidermal growth factor receptor (EGFR) signaling. After permanent focal cerebral ischemia, tPA knockout mice developed more severe sensorimotor and cognitive deficits and greater axonal and myelin injury than wild-type mice, suggesting that endogenously expressed tPA promotes long-term neurological recovery after stroke. In tPA knockout mice, intranasal administration of recombinant tPA protein 6 hours poststroke and 7 more times at 2 d intervals mitigated white matter injury, improved axonal conduction, and enhanced neurological recovery. Consistent with the proaxonal growth effects observed in vitro, exogenous tPA delivery increased poststroke axonal sprouting of corticobulbar and corticospinal tracts, which might have contributed to restoration of neurological functions. Notably, recombinant mutant tPA-S478A lacking protease activity (but retaining the EGF-like domain) was as effective as wild-type tPA in rescuing neurological functions in tPA knockout stroke mice. These findings demonstrate that tPA improves long-term functional outcomes in a clinically relevant stroke model, likely by promoting brain plasticity through EGFR signaling. Therefore, treatment with the protease-dead recombinant tPA-S478A holds particular promise as a neurorestorative therapy, as the risk for triggering intracranial hemorrhage is eliminated and tPA-S478A can be delivered intranasally hours after stroke.