New insights into the roles of oligodendrocytes regulation in ischemic stroke recovery.

Oligodendrocytes (OLs), the myelin-forming cells of the central nervous system, are integral to axonal integrity and function. Hypoxia-ischemia episodes can cause severe damage to these vulnerable cells through excitotoxicity, oxidative stress, inflammation, and mitochondrial dysfunction, leading to axonal dystrophy, neuronal dysfunction, and neurological impairments. OLs damage can result in demyelination and myelination disorders, severely impacting axonal function, structure, metabolism, and survival. Adult-onset stroke, periventricular leukomalacia, and post-stroke cognitive impairment primarily target OLs, making them a critical therapeutic target. Therapeutic strategies targeting OLs, myelin, and their receptors should be given more emphasis to attenuate ischemia injury and establish functional recovery after stroke. This review summarizes recent advances on the function of OLs in ischemic injury, as well as the present and emerging principles that serve as the foundation for protective strategies against OLs deaths.

Association of admission neutrophil serine proteinases levels with the outcomes of acute ischemic stroke: a prospective cohort study.

BACKGROUND: Neutrophil serine proteinases (NSPs), released by activated neutrophils, are key proteins involved in the pathophysiologic processes of stroke. NSPs are also implicated in the process and response of thrombolysis. This study aimed to analyze three NSPs (neutrophil elastase, cathepsin G, and proteinase 3) in relation to acute ischemic stroke (AIS) outcomes and in relation to the outcomes of patients treated with intravenous recombinant tissue plasminogen activator (IV-rtPA). METHODS: Among 736 patients prospectively recruited at the stroke center from 2018 to 2019, 342 patients diagnosed with confirmed AIS were included. Plasma neutrophil elastase (NE), cathepsin G (CTSG), and proteinase 3 (PR3) concentrations were measured on admission. The primary endpoint was unfavorable outcome defined as modified Rankin Scale score 3-6 at 3 months, and the secondary endpoints were symptomatic intracerebral hemorrhage (sICH) within 48 h, and mortality within 3 months. In the subgroup of patients who received IV-rtPA, post-thrombolysis early neurological improvement (ENI) (defined as National Institutes of Health Stroke Scale score = 0 or decrease of ≥ 4 within 24 h after thrombolysis) was also included as the secondary endpoint. Univariate and multivariate logistic regression analyses were performed to evaluate the association between NSPs levels and AIS outcomes. RESULTS: Higher NE and PR3 plasma levels were associated with the 3-month mortality and 3-month unfavorable outcome. Higher NE plasma levels were also associated with the risk of sICH after AIS. After adjusting for potential confounders, plasma NE level > 229.56 ng/mL (odds ratio [OR] = 4.478 [2.344-8.554]) and PR3 > 388.77 ng/mL (OR = 2.805 [1.504-5.231]) independently predicted the 3-month unfavorable outcome. Regarding rtPA treatment, patients with NE plasma concentration > 177.22 ng/mL (OR = 8.931 [2.330-34.238]) or PR3 > 388.77 ng/mL (OR = 4.275 [1.045-17.491]) were over 4 times more likely to suffer unfavorable outcomes after rtPA treatment. The addition of NE and PR3 to clinical predictors of unfavorable functional outcome after AIS and the outcome after rtPA treatment improved discrimination as well as reclassification (integrated discrimination improvement = 8.2% and 18.1%, continuous net reclassification improvement = 100.0% and 91.8%, respectively). CONCLUSIONS: Plasma NE and PR3 are novel and independent predictors of 3-month functional outcomes after AIS. Plasma NE and PR3 also possess predictive value to identify patients with unfavorable outcomes after rtPA treatment. NE is probably an important mediator of the effects of neutrophils on stroke outcomes, which worth further investigation.

LC-MS/MS metabolomic profiling of the protective butylphthalide effect in cerebral ischemia/reperfusion mice.

OBJECTIVES: This study was designed to investigate metabolic biomarker changes and related metabolic pathways of Butylphthalide (NBP) on cerebral ischemia/reperfusion. METHODS: In this study, a mouse cerebral ischemia/reperfusion (I/R) model was prepared using the middle cerebral artery occlusion method, and neurobehavioral score and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining experiments were used to confirm the obvious NBP anti-cerebral ischemia effect. The protective effect of NBP in the mouse cerebral I/R model and its metabolic pathway and mechanism were investigated using mouse blood samples. RESULTS: The metabolic profiles of mice in the I/R+NBP, I/R, and sham groups were significantly different. Under the condition that I/R vs. sham was downregulated and I/R + NBP vs. I/R was upregulated, 88 differential metabolites, including estradiol, ubiquinone-2, 2-oxoarginine, and L-histidine trimethylbetaine, were screened and identified. The related metabolic pathways involved arginine and proline metabolism, oxidative phosphorylation, ubiquitin and other terpenoid-quinone biosynthesis, and estrogen signaling. CONCLUSIONS: Metabolomics was used to elucidate the NBP mechanism in cerebral ischemia treatment in mice, revealing synergistic NBP pharmacological characteristics with multiple targets.

Intravenous antagomiR-494 lessens brain-infiltrating neutrophils by increasing HDAC2-mediated repression of multiple MMPs in experimental stroke.

Neutrophil infiltration and phenotypic transformation are believed to contribute to neuronal damage in ischemic stroke. Emerging evidence suggests that histone deacetylase 2 (HDAC2) is an epigenetic regulator of inflammatory cells. Here, we aimed to investigate whether microRNA-494 (miR-494) affects HDAC2-mediated neutrophil infiltration and phenotypic shift. MiR-494 levels in neutrophils from acute ischemic stroke (AIS) patients were detected by real-time PCR. Chromatin Immunoprecipitation (ChIP)-Seq was performed to clarify which genes are the binding targets of HDAC2. Endothelial cells and cortical neurons were subjected to oxygen-glucose deprivation (OGD), transwell assay was conducted to examine neutrophil migration through endothelial cells, and neuronal injury was examined after stimulating with supernatant from antagomiR-494-treated neutrophils. C57BL/6J mice were subjected to transient middle cerebral artery occlusion (MCAO) and antagomiR-494 was injected through tail vein immediately after reperfusion, and neutrophil infiltration and phenotypic shift was examined. We found that the expression of miR-494 in neutrophils was significantly increased in AIS patients. HDAC2 targeted multiple matrix metalloproteinases (MMPs) and Fc-gamma receptor III (CD16) genes in neutrophils of AIS patients. Furthermore, antagomiR-494 repressed expression of multiple MMPs genes, including MMP7, MMP10, MMP13, and MMP16, which reduced the number of brain-infiltrating neutrophils by regulating HDAC2. AntagomiR-494 could also exert its neuroprotective role through inhibiting the shift of neutrophils toward pro-inflammatory N1 phenotype in vivo and in vitro. Taken together, miR-494 may serve as an alternative predictive biomarker of the outcome of AIS patients, and antagomiR-494 treatment decreases the expression of multiple MMPs and the infiltration of neutrophils and inhibits the shift of neutrophils into N1 phenotype partly by targeting HDAC2.

Tunable coffee-ring formation of halloysite nanotubes by evaporating sessile drops.

Halloysite nanotubes (HNTs) are one-dimensional clay nanomaterials with a length of 200-1000 nm and a diameter of ∼50 nm. Understanding the self-assembly behavior of such unique nanoparticles is important to develop their applications in functional devices. In this study, the "coffee-ring" patterns of HNTs are investigated which are formed by evaporation of the sessile droplets of HNT aqueous dispersion on different substrates. The coffee-ring pattern with various dimensions was characterized using a polarizing microscope (POM), a scanning electron microscope (SEM), and a 3D optical profilometer. The diameter, height, and area of the coffee-ring patterns depend on the concentration of HNT dispersion, the droplet volume, and surface wettability. POM and SEM results suggested that the nanotubes were highly ordered in the edge and the middle of the coffee-ring. The coffee-ring effect of HNTs could be suppressed by increasing the evaporation temperature of substrates or adding polymer additives. In addition, multiple-ring patterns consistent with protein rings surrounding HNT rings were formed, which can be utilized to detect the presence of proteins in biological samples. This work illustrated the relationship between the formation of coffee-ring patterns and the experimental conditions, which provided an additional research chance and allowed application development for HNTs using the liquid droplet self-assembly.

sLOX-1: A Molecule for Evaluating the Prognosis of Recurrent Ischemic Stroke.

Several clinical parameters and biomarkers have been proposed as prognostic markers for stroke. However, it has not been clarified whether the risk factors affecting the prognosis of patients with recurrent and first-ever stroke are similar. In this study, we aimed to explore the relationship between soluble lectin-like oxidized low-density lipoprotein receptor 1 (sLOX-1) levels and the prediction of the functional outcome in patients with recurrent and first-ever stroke. A total of 266 patients with recurrent and first-ever stroke, who underwent follow-up for 3 months, were included in this study. Plasma samples were collected within 24 h after onset. The results showed that biomarkers for the prognosis of patients with recurrent stroke were different from that of those with first-ever stroke. sLOX-1 levels were correlated with modified Rankin Scale scores of patients with recurrent stroke alone (r = 0.3232, p = 0.001). sLOX-1 levels were also associated with an increased risk of unfavorable outcomes in patients with recurrent stroke with an adjusted odds ratio of 1.489 (95% confidence interval, 1.204-1.842, p < 0.0001). Combining the risk factors showed greater accuracy for prognosis, yielding a sensitivity of 93.2% and a specificity of 75%, with an area under the curve of 0.916, evaluated by the receiver operating characteristic curve. These findings suggest that the diagnosis and prognosis are different between patients with recurrent stroke and those with first-ever stroke, and sLOX-1 level is an independent prognostic marker in patients with recurrent stroke.

Prognostic significance of plasma IL-2 and sIL-2Rα in patients with first-ever ischaemic stroke.

BACKGROUND: An imbalance between circulating neuroprotective and neurotoxic T cell subsets leads to poor prognosis in acute ischaemic stroke (AIS). Preclinical studies have indicated that the soluble form of the interleukin-2 receptor α (sIL-2Rα)-IL-2 complex regulates T cell differentiation. However, the association between sIL-2Rα levels and AIS remains unclear. METHODS: A total of 201 first-ever AIS patients within 24 h after stroke onset and 76 control subjects were recruited. The National Institutes of Health Stroke Scale (NIHSS) score and 3-month functional outcome (modified Rankin Scale [mRS] score) at admission were assessed. Plasma sIL-2Rα and IL-2 levels at admission were measured. Prognostic significance was identified by using univariate and multivariate logistic regression analyses. RESULTS: Patients with poor functional outcomes at 3 months had significantly higher levels of sIL-2Rα and lower levels of IL-2 than patients with good outcomes. Moreover, sIL-2Rα levels showed a strong positive correlation with NIHSS and mRS scores (p < 0.0001), whereas IL-2 levels were negatively correlated with mRS scores (p < 0.01). Univariate analyses showed that higher sIL-2Rα and IL-2 levels were associated with an increased and reduced risk of unfavourable outcomes, respectively. After adjusting for confounding variables, the sIL-2Rα level remained independently associated with an increased risk of an unfavourable outcome, and adding sIL-2Rα levels to the conventional risk factor model significantly improved risk reclassification (net reclassification improvement 17.56%, p = 0.003; integrated discrimination improvement 5.78%, p = 0.0003). CONCLUSIONS: sIL-2Rα levels represent a novel, independent prognostic marker that can improve the currently used risk stratification of AIS patients. Our findings also highlight that elevated plasma sIL-2Rα and IL-2 levels manifested opposite correlations with functional outcome, underlining the importance of IL-2/IL-2R autocrine loops in AIS.

The effects of erythropoietin on neurogenesis after ischemic stroke.

Erythropoietin has been researched for its neuroprotective effects in ischemic stroke for over 30 years. Although erythropoietin can cause side effects that need to be controlled, it has been suggested to be effective in enhancing the prognosis of patients who are out of the therapeutic time window and have not received recombinant tissue plasminogen activator therapy. Studies on the mechanism of the function of erythropoietin have shown that it has various protective effects in ischemic brain injury after stroke, including promoting neurogenesis. In this review, we discuss the effects of erythropoietin on neurogenesis after ischemic brain injury and provide references for effective treatments for ischemic stroke, which is one of the leading causes of death worldwide.

The Chinese herb Fructus Broussonetiae aids learning and memory in chronic cerebral hypoperfusion by reducing proinflammatory microglia activation in rats.

The neuroprotective role of Fructus Broussonetiae in a model of chronic cerebral hypoperfusion with cognitive decline was focused on neural plasticity and microglia/macrophage polarization. Chronic cerebral hypoperfusion was induced by bilateral common carotid artery ligation. Fructus Broussonetiae shortened escape latency and added the number of platform crossings of rats, up-regulated the expression of synaptophysin in the gray matter and increased myelin basic protein expression in the white matter. Further mechanistic experiments were conducted to examine microglia activation and M1/M2 polarization. It was shown that Fructus Broussonetiae reduced the activation of microglia revealed by decreased expression of ionized calcium-binding adapter molecule-1, inhibited M1 polarization of microglia and improved microglial M2 polarization shown by down-regulated the expression of inducible nitric oxide synthase and Fc fragment of IgG receptor IIIa and up-regulated the expression of arginase-1. In conclusion, the Chinese herb Fructus Broussonetiae can improve cognitive function following chronic cerebral hypoperfusion by down-regulating the activation of microglia, inhibiting microglial M1 polarization, and improving neural plasticity.

Synergistic Interaction Between Zinc and Reactive Oxygen Species Amplifies Ischemic Brain Injury in Rats.

Background and Purpose- Although intracellular zinc accumulation has been shown to contribute to neuronal death after cerebral ischemia, the mechanism by which zinc keeps on accumulating to cause severe brain damage remains unclear. Herein the dynamic cause-effect relationships between zinc accumulation and reactive oxygen species (ROS) production during cerebral ischemia/reperfusion are investigated. Methods- Rats were treated with zinc chelator, ROS scavenger, mitochondria-targeted ROS inhibitor, or NADPH oxidase inhibitor during a 90-minute middle cerebral artery occlusion. Cytosolic labile zinc, ROS level, cerebral infarct volume, and neurological functions were assessed after ischemia/reperfusion. Results- Zinc and ROS were colocalized in neurons, leading to neuronal apoptotic death. Chelating zinc reduced ROS production at 6 and 24 hours after reperfusion, whereas eliminating ROS reduced zinc accumulation only at 24 hours. Furthermore, suppression of mitochondrial ROS production reduced the total ROS level and brain damage at 6 hours after reperfusion but did not change zinc accumulation, indicating that ROS is produced mainly from mitochondria during early reperfusion and the initial zinc release is upstream of ROS generation after ischemia. Suppression of NADPH oxidase decreased ROS generation, zinc accumulation, and brain damage only at 24 hours after reperfusion, indicating that the majority of ROS is produced by NADPH oxidase at later reperfusion time. Conclusions- This study provides the direct evidence that there exists a positive feedback loop between zinc accumulation and NADPH oxidase-induced ROS production, which greatly amplifies the damaging effects of both. These findings reveal that different ROS-generating source contributes to ischemia-generated ROS at different time, underscoring the critical importance of spatial and temporal factors in the interaction between ROS and zinc accumulation, and the consequent brain injury, after cerebral ischemia/reperfusion.

Diabetes Mellitus Impairs White Matter Repair and Long-Term Functional Deficits After Cerebral Ischemia.

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 (WM) impairment, but the underlying mechanism is not completely understood. This study was designed to test the hypothesis that T2DM impedes poststroke WM 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, WM damage, oligodendrogenesis, and microglia/macrophage responses were evaluated up to 35 days after stroke. The functional integrity of WM was measured by electrophysiology. Primary microglia-oligodendrocyte cocultures were used for additional mechanistic studies. Results- T2DM exacerbated structural damage and impaired conduction of compound action potentials in WM 35 days after stroke. The deterioration in WM integrity correlated with poor sensorimotor performance. Furthermore, T2DM impaired the proliferation of oligodendrocyte precursor cells and the generation of new myelinating oligodendrocytes. T2DM also promoted a shift of microglia/macrophage phenotype toward the proinflammatory modality. Coculture studies confirmed that microglia/macrophage polarization toward the proinflammatory phenotype under high glucose conditions suppressed oligodendrocyte precursor cell differentiation. Conclusions- Deterioration of WM 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 proinflammatory phenotype, significantly impairing oligodendrocyte precursor cell differentiation and WM repair.

Chrysophanol attenuates nitrosative/oxidative stress injury in a mouse model of focal cerebral ischemia/reperfusion.

Nitrosative/oxidative stress plays an important role in neuronal death following cerebral ischemia/reperfusion (I/R). Chrysophanol (CHR) has been shown to afford significant neuroprotection on ischemic stroke, however, whether its mechanism is related to attenuating nitrosative/oxidative stress is not clear. In the present study, we investigated the effect of CHR on neuronal injury related to nitric oxide (NO) production by using mouse middle cerebral artery occlusion (MCAO) model. Our results revealed that nitrite plus nitrate (NOx-) and 3-nitrotyrosine (3-NT) levels increased in ischemic brain 14 days after reperfusion, and were subsequently attenuated by CHR treatment. Moreover, 3-NT is colocalized with NeuN and TUNEL, suggesting that neuronal apoptosis following I/R is associated with 3-NT and CHR suppresses NO-associated neuronal cell death. Accordingly, cleaved caspase-3 expression in ischemic brain was decreased by CHR treatment. I/R also decreased the activity of total superoxide dismutase (SOD) and manganese-dependent SOD (MnSOD), whilst increased reactive oxygen species (ROS) production significantly. Interestingly, CHR reversed this decrease in total SOD, and MnSOD activity, and inhibited ROS generation in the ischemic brain. Taken together, our results provide direct evidence suggesting that CHR attenuates nitrosative/oxidative stress injury induced by I/R, providing a novel therapeutic target in the treatment of acute ischemic stroke.

L-3-n-Butylphthalide reduces ischemic stroke injury and increases M2 microglial polarization.

Overwhelming evidence suggests that microglia play an important role in ischemic injury and they polarize into two different phenotypes with distinct functions after ischemic stroke. We performed the present study to investigate whether L-3-n butylphthalide (NBP) has an effect on microglial polarization. Mice were subjected to transient middle cerebral artery occlusion (MCAO) for 45 min, and then immediately after reperfusion were treated with NBP or vehicle via the caudal vein for 7 consecutive days. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed that NBP treatment resulted in a tendency to decrease cerebral infarct volume at 1 day after MCAO, and significant decreased infarct volume at 3 days after MCAO. Sensorimotor function was evaluated by the adhesive removal test and balance beam test, which were superior in NBP-treated mice compared with vehicle-treated mice at 1 and 3 days after MCAO. Immunofluorescent staining further indicated that NBP treatment significantly increased the number of CD206+/Iba1+ M2 microglia/macrophages and reduced the number of CD16+/Iba1+ M1 cells at 3 and 7 days after MCAO reperfusion. Western blot also showed an elevation of M2 marker (arginase-1) in NBP-treated brains at 7 days after MCAO. In conclusion, our results clearly show that NBP treatment significantly mitigates ischemic brain damage and promotes recovery of neurological function in early phase after ischemic stroke, probably by skewing M1 microglia/macrophages polarization towards M2 phenotype. Thus, our study provides new evidence that NBP might be a promising candidate for ameliorating injury caused by ischemic stroke.

Long Noncoding RNA H19 Promotes Neuroinflammation in Ischemic Stroke by Driving Histone Deacetylase 1-Dependent M1 Microglial Polarization.

BACKGROUND AND PURPOSE: Long noncoding RNA H19 is repressed after birth, but can be induced by hypoxia. We aim to investigate the impact on and underlying mechanism of H19 induction after ischemic stroke. METHODS: Circulating H19 levels in stroke patients and mice subjected to middle cerebral artery occlusion were assessed using real-time polymerase chain reaction. H19 siRNA and histone deacetylase 1 (HDAC1) plasmid were used to knock down H19 and overexpress HDAC1, respectively. Microglial polarization and ischemic outcomes were assessed in middle cerebral artery occlusion mice and BV2 microglial cells subjected to oxygen-glucose deprivation. RESULTS: Circulating H19 levels were significantly higher in stroke patients compared with healthy controls, indicating high diagnostic sensitivity and specificity. Moreover, plasma H19 levels showed a positive correlation with National Institute of Health Stroke Scale score and tumor necrosis factor-α levels. After middle cerebral artery occlusion in mice, H19 levels increased in plasma, white blood cells, and brain. Intracerebroventricular injection of H19 siRNA reduced infarct volume and brain edema, decreased tumor necrosis factor-α and interleukin-1ß levels in brain tissue and plasma, and increased plasma interleukin-10 concentrations 24 hours poststroke. Additionally, H19 knockdown attenuated brain tissue loss and neurological deficits 14 days poststroke. BV2 cell-based experiments showed that H19 knockdown blocked oxygen-glucose deprivation-driven M1 microglial polarization, decreased production of tumor necrosis factor-α and CD11b, and increased the expression of Arg-1 and CD206. Furthermore, H19 knockdown reversed oxygen-glucose deprivation-induced upregulation of HDAC1 and downregulation of acetyl-histone H3 and acetyl-histone H4. In contrast, HDAC1 overexpression negated the effects of H19 knockdown. CONCLUSIONS: Our findings indicate that H19 promotes neuroinflammation by driving HDAC1-dependent M1 microglial polarization, suggesting a novel H19-based diagnosis and therapy for ischemic stroke.

Zinc contributes to acute cerebral ischemia-induced blood-brain barrier disruption.

Zinc ions are stored in synaptic vesicles and cerebral ischemia triggers their release from the terminals of neurons. Zinc accumulation in neurons has been shown to play an important role in neuronal death following ischemia. However, almost nothing is known about whether zinc is involved in ischemia-induced blood-brain barrier (BBB) disruption. Herein, we investigated the contribution of zinc to ischemia-induced acute BBB disruption and the possible molecular mechanisms using both cellular and animal models of cerebral ischemia. Zinc greatly increased BBB permeability and exacerbated the loss of tight junction proteins (Occludin and Claudin-5) in the endothelial monolayer under oxygen glucose deprivation conditions. In cerebral ischemic rats, a dramatically elevated level of zinc accumulation in microvessels themselves was observed in isolated microvessels and in situ, showing the direct interaction of zinc on ischemic microvessels. Treatment with a specific zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), even at 60-min post-ischemia onset, could greatly attenuate BBB permeability in the ischemic rats as measured by Evan's Blue extravasation, edema volume and magnetic resonance imaging. Furthermore, zinc accumulation in microvessels activated the superoxide/matrix metalloproteinase-9/-2 pathway, which leads to the loss of tight junction proteins (Occludin and Claudin-5) and death of endothelial cells in microvessels themselves. Our findings reveal a novel mechanism of cerebral ischemia-induced BBB damage, and implicate zinc as an effective and viable new target for reducing acute BBB damage following ischemic stroke.

Overexpression of iASPP-SV in glioma is associated with poor prognosis by promoting cell viability and antagonizing apoptosis.

Inhibitor of apoptosis-stimulating protein of p53 (iASPP), encoded by PPP1R13L gene, is often overexpressed in human cancers. From the PPP1R13L gene, at least two isoforms, iASPP-L and iASPP-SV, are produced through alternative splicing. However, the role of these isoforms in glioma is still elusive. In this study, we examined the expression of iASPP-SV in astrocytic glioma tissues with different grades and normal human cerebral tissues. The result showed a higher messenger RNA (mRNA) expression level of iASPP-SV in astrocytic glioma patients with World Health Organization (WHO) grade II to IV in comparison to the normal controls. Additionally, mRNA expression level of iASPP-SV was gradually increased with the raise of the grade in glioma. High mRNA expression level of iASPP-SV was significantly associated with malignant WHO grades (P < 0.001). The protein expression level of iASPP-SV was consistent with the mRNA expression level. The Kaplan-Meier analysis revealed that high iASPP-SV mRNA expression significantly affected overall survival and progression-free survival (both P < 0.001). Furthermore, multivariate analysis indicated that the mRNA expression of iASPP-SV was an independent prognostic marker in glioma (P < 0.001). To further explore the role of iASPP-SV in glioma, U87 cells were transfected with iASPP-SV by lentivirus and then treated with temozolomide (TMZ). Overexpression of iASPP-SV promoted the cell viability and downregulated the expression of pro-apoptosis genes (Bax, Puma, p21, and Noxa) to inhibit apoptosis induced by TMZ. Our study provides the first evidence that high iASPP-SV expression may be a novel prognostic factor and therapeutic target for glioma.

MicroRNA-424 protects against focal cerebral ischemia and reperfusion injury in mice by suppressing oxidative stress.

BACKGROUND AND PURPOSE: We previously showed that the microRNA miR-424 protects against permanent cerebral ischemic injury in mice by suppressing microglia activation. This study investigated the role of miR-424 in transient cerebral ischemia in mice with a focus on oxidative stress-induced neuronal injury. METHODS: Transient cerebral ischemia was induced in C57/BL6 mice by middle cerebral artery occlusion for 1 hour followed by reperfusion (ischemia/reperfusion). The miR-424 level in the peri-infarct cortex was quantified. Mice were also administered miR-424 angomir by intracerebroventricular injection. Cerebral infarct volume, neuronal apoptosis, and levels of oxidative stress markers and antioxidants were evaluated. In an in vitro experiment, primary cortical neurons were exposed to H2O2 and treated with miR-424 angomir, nuclear factor erythroid 2-related factor 2 siRNA, and superoxide dismutase (SOD) inhibitor; cell activity, lactate dehydrogenase release, malondialdehyde level, and manganese (Mn)SOD activity were then evaluated. RESULTS: MiR-424 levels in the peri-infarct cortex increased at 1 and 4 hours then decreased 24 hours after reperfusion. Treatment with miR-424 decreased infarct volume and inhibited neuronal apoptosis after ischemia/reperfusion, reduced reactive oxygen species and malondialdehyde levels in the cortex, and increased the expression and activation of MnSOD as well as the expression of extracellular SOD and the redox-sensitive transcription factor nuclear factor erythroid 2-related factor. In neuronal cultures, miR-424 treatment abrogated H2O2-induced injury, as evidenced by decreased lactate dehydrogenase leakage and malondialdehyde level and increased cell viability and MnSOD activity; the protective effects of miR-424 against oxidative stress were reversed by nuclear factor erythroid 2-related factor knockdown and SOD inhibitor treatment. CONCLUSIONS: MiR-424 protects against transient cerebral ischemia/reperfusion injury by inhibiting oxidative stress.

Normobaric hyperoxia slows blood-brain barrier damage and expands the therapeutic time window for tissue-type plasminogen activator treatment in cerebral ischemia.

BACKGROUND AND PURPOSE: Prolonged ischemia causes blood-brain barrier (BBB) damage and increases the incidence of neurovasculature complications secondary to reperfusion. Therefore, targeting ischemic BBB damage pathogenesis is critical to reducing neurovasculature complications and expanding the therapeutic time window of tissue-type plasminogen activator (tPA) thrombolysis. This study investigates whether increasing cerebral tissue PO2 through normobaric hyperoxia (NBO) treatment will slow the progression of BBB damage and, thus, improve the outcome of delayed tPA treatment after cerebral ischemia. METHODS: Rats were exposed to NBO (100% O2) or normoxia (21% O2) during 3-, 5-, or, 7-hour middle cerebral artery occlusion. Fifteen minutes before reperfusion, tPA was continuously infused to rats for 30 minutes. Neurological score, mortality rate, and BBB permeability were determined. Matrix metalloproteinase-9 was measured by gelatin zymography and tight junction proteins (occludin and cluadin-5) by Western blot in the isolated cerebral microvessels. RESULTS: NBO slowed the progression of ischemic BBB damage pathogenesis, evidenced by reduced Evan blue leakage, smaller edema, and hemorrhagic volume in NBO-treated rats. NBO treatment reduced matrix metalloproteinase-9 induction and the loss of tight junction proteins in ischemic cerebral microvessels. NBO-afforded BBB protection was maintained during tPA reperfusion, resulting in improved neurological functions, significant reductions in brain edema, hemorrhagic volume, and mortality rate, even when tPA was given after prolonged ischemia (7 hours). CONCLUSIONS: Early NBO treatment slows ischemic BBB damage pathogenesis and significantly improves the outcome of delayed tPA treatment, providing new evidence supporting NBO as an effective adjunctive therapy to extend the time window of tPA thrombolysis for ischemic stroke.

Intra-artery infusion of recombinant human erythropoietin reduces blood-brain barrier disruption in rats following cerebral ischemia and reperfusion.

OBJECTIVES: Intra-artery infusion of recombinant human erythropoietin (rhEPO) has recently been reported to confer neuroprotection against cerebral ischemia-reperfusion injury in animal models; however, the molecular mechanisms are still under investigation. The present study focused on the specific mechanism involved in blood-brain barrier (BBB) disruption. METHODS: Thirty-six male and nine female Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion to induce focal cerebral ischemia, and administrated rhEPO at a dose of 800 U/kg through MCA infusion at the beginning of reperfusion. Neurobehavioral deficits, brain edema, and infarct volume were evaluated after 2 h of ischemia and 24 h of reperfusion. BBB permeability was assessed by quantifying the extravasation of Evans blue (EB) dye. The expression of tight junction proteins and matrix metalloproteinases (MMPs) (Claudin-5, Occludin, MMP-2, and MMP-9) in microvessels were detected by immunofluorescence and western blot. The activities of MMPs in the cerebral microvessels were determined by gelatin zymography. RESULTS: Treatment with rhEPO through the MCA strongly alleviated infarct volume, brain edema, and improved neurobehavioral outcomes in male and female rats. In addition, rhEPO remarkably suppressed the EB extravasation induced by brain ischemia. Furthermore, rhEPO prevented degradation of Claudin-5 and Occludin, and reduced the expression and activity of MMP-2 and MMP-9 in isolated brain microvessels. CONCLUSIONS: Treatment with rhEPO through MCA infusion prevented brain edema formation and infarction through inhibition of MMP-mediated BBB disruption in acute ischemic stroke.

Chelating intracellularly accumulated zinc decreased ischemic brain injury through reducing neuronal apoptotic death.

BACKGROUND AND PURPOSE: Zinc has been reported to possess both neurotoxic and neuroprotective capabilities. The effects of elevated intracellular zinc accumulation following transient focal cerebral ischemia remain to be fully elucidated. Here, we investigated whether removing zinc with the membrane-permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), would decrease the intracellular levels of zinc in the ischemic tissue, leading to reduced brain damage and improved neurological outcomes. METHODS: Rats were pretreated with TPEN or vehicle before or after a 90-minute middle cerebral artery occlusion. Cerebral infarct volume, neurological functions, neuronal apoptosis, poly(ADP-ribose) polymerase activity, and cytosolic labile zinc were assessed after ischemia and reperfusion. RESULTS: Cerebral ischemia caused a dramatic cytosolic labile zinc accumulation in the ischemic tissue, which was decreased markedly by TPEN (15 mg/kg) pretreatment. Chelating zinc lead to reduced infarct volume compared with vehicle-treated middle cerebral artery occlusion rats, accompanied by much improved neurological assessment and motor function, which were sustained for 14 days after reperfusion. We also determined that reducing zinc accumulation rescued neurons from ischemia-induced apoptotic death by reducing poly(ADP-ribose) polymerase-1 activation. CONCLUSIONS: Ischemia-induced high accumulation of intracellular zinc significantly contributed to ischemic brain damage through promotion of neuronal apoptotic death. Removing zinc may be an effective and novel approach to reduce ischemic brain injury.