Neat1 decreases neuronal apoptosis after oxygen and glucose deprivation.

Studies have shown that downregulation of nuclear-enriched autosomal transcript 1 (Neat1) may adversely affect the recovery of nerve function and the increased loss of hippocampal neurons in mice. Whether Neat1 has protective or inhibitory effects on neuronal cell apoptosis after secondary brain injury remains unclear. Therefore, the effects of Neat1 on neuronal apoptosis were observed. C57BL/6 primary neurons were obtained from the cortices of newborn mice and cultured in vitro, and an oxygen and glucose deprivation cell model was established to simulate the secondary brain injury that occurs after traumatic brain injury in vitro. The level of Neat1 expression in neuronal cells was regulated by constructing a recombinant adenovirus to infect neurons, and the effects of Neat1 expression on neuronal apoptosis after oxygen and glucose deprivation were observed. The experiment was divided into four groups: the control group, without any treatment, received normal culture; the oxygen and glucose deprivation group were subjected to the oxygen and glucose deprivation model protocol; the Neat1 overexpression and Neat1 downregulation groups were treated with Neat1 expression intervention techniques and were subjected to the in oxygen and glucose deprivation protocol. The protein expression levels of neurons p53-induced death domain protein 1 (PIDD1, a pro-apoptotic protein), caspase-2 (an apoptotic priming protein), cytochrome C (a pro-apoptotic protein), and cleaved caspase-3 (an apoptotic executive protein) were measured in each group using the western blot assay. To observe changes in the intracellular distribution of cytochrome C, the expression levels of cytochrome C in the cytoplasm and mitochondria of neurons from each group were detected by western blot assay. Differences in the cell viability and apoptosis rate between groups were detected by cell-counting kit 8 assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, respectively. The results showed that the apoptosis rate, PIDD1, caspase-2, and cleaved caspase-3 expression levels significantly decreased, and cell viability significantly improved in the Neat1 overexpression group compared with the oxygen and glucose deprivation group; however, Neat1 downregulation reversed these changes. Compared with the Neat1 downregulation group, the cytosolic cytochrome C level in the Neat1 overexpression group significantly decreased, and the mitochondrial cytochrome C level significantly increased. These data indicate that Neat1 upregulation can reduce the release of cytochrome C from the mitochondria to the cytoplasm by inhibiting the PIDD1-caspase-2 pathway, reducing the activation of caspase-3, and preventing neuronal apoptosis after oxygen and glucose deprivation, which might reduce secondary brain injury after traumatic brain injury. All experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China, on December 19, 2020 (approval No. 2020-895).

Downregulation of miR-491-5p promotes neovascularization after traumatic brain injury.

MicroRNA-491-5p (miR-491-5p) plays an important role in regulating cell proliferation and migration; however, the effect of miR-491-5p on neovascularization after traumatic brain injury remains poorly understood. In this study, a controlled cortical injury model in C57BL/6 mice and an oxygen-glucose deprivation model in microvascular endothelial cells derived from mouse brain were established to simulate traumatic brain injury in vivo and in vitro, respectively. In the in vivo model, quantitative real-time-polymerase chain reaction results showed that the expression of miR-491-5p increased or decreased following the intracerebroventricular injection of an miR-491-5p agomir or antagomir, respectively, and the expression of miR-491-5p decreased slightly after traumatic brain injury. To detect the neuroprotective effects of miR-491-p, neurological severity scores, Morris water maze test, laser speckle techniques, and immunofluorescence staining were assessed, and the results revealed that miR-491-5p downregulation alleviated neurological dysfunction, promoted the recovery of regional cerebral blood flow, increased the number of lectin-stained microvessels, and increased the survival of neurons after traumatic brain injury. During the in vitro experiments, the potential mechanism of miR-491-5p on neovascularization was explored through quantitative real-time-polymerase chain reaction, which showed that miR-491-5p expression increased or decreased in brain microvascular endothelial cells after transfection with an miR-491-5p mimic or inhibitor, respectively. Dual-luciferase reporter and western blot assays verified that metallothionein-2 was a target gene for miR-491-5p. Cell counting kit 8 (CCK-8) assay, flow cytometry, and 2?,7?-dichlorofluorescein diacetate (DCFH-DA) assay results confirmed that the downregulation of miR-491-5p increased brain microvascular endothelial cell viability, reduced cell apoptosis, and alleviated oxidative stress under oxygen-glucose deprivation conditions. Cell scratch assay, Transwell assay, tube formation assay, and western blot assay results demonstrated that miR-491-5p downregulation promoted the migration, proliferation, and tube formation of brain microvascular endothelial cells through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor pathway. These findings confirmed that miR-491-5p downregulation promotes neovascularization, restores cerebral blood flow, and improves the recovery of neurological function after traumatic brain injury. The mechanism may be mediated through a metallothionein-2-dependent hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway and the alleviation of oxidative stress. All procedures were approved by Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, China (approval No. 2020-304) on June 22, 2020.

Clinical study of changes of cerebral microcirculation in cerebral vasospasm after SAH.

Aim to investigate the changes of cerebral microcirculation after subarachnoid hemorrhage (SAH) and its association with cerebral vasospasm (CVS) after SAH. CTP was performed in 85 patients with SAH and 35 controls. Cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) were recorded for final analysis. CTP parameters were compared between (1) SAH group and control group, (2) CVS group and non-CVS group (nCVS), (3) symptomatic CVS (sCVS) group and asymptomatic CVS (asCVS) group. Compared to control group, there were significant differences in CBF and MTT of SAH patients (P<0.05). Among SAH patients, the CBF and MTT (a decreased CBF and a prolonged MTT) of CVS patients were significantly different from those of non-CVS patients (P<0.05). In 46 CVS patients, sCVS group presented significantly lower CBF and more prolonged MTT than asCVS patients (P<0.05). Seven cases with MTT between 6.31 and 12.72 s showed delayed ischemic neurological deficit (DIND), two of whom had hemiplegia, and one died. Our findings suggest that CTP examination contributes to uncover the changes of cerebral microcirculation after SAH, and the changes of cerebral microcirculation are associated with CVS post SAH.

Delayed intracranial hemorrhage associated with antiplatelet therapy in stent-assisted coil embolized cerebral aneurysms.

Administration of oral clopidogrel plus aspirin is the most important regimen to reduce thromboembolic complications in stent-assisted coil embolization of cerebral aneurysm. However, such therapy may increase the risk of hemorrhage. The purpose of this study is to analyze the effect of two different antiplatelet regimens on hemorrhagic and thromboembolic complication rates around the stent-assisted coil embolization period. Records over a 2-year period were reviewed in a retrospective cohort study. For 49 consecutive stent-assisted coil embolization procedures over 41 patients, nine patients received routine antiplatelet drugs (300 mg aspirin and 75 mg clopidogrel) for 3 days before embolization, and 32 received a loading dose of antiplatelet drugs (300 mg aspirin and 300 mg clopidogrel) just before induction of anesthesia. Delayed intracerebral hemorrhage (DIH) was observed more often in the routine antiplatelet group (2/9 cases, 22.2%) in comparison with the loading group (0/32 cases, 0%; P = 0.044; Fisher exact test). The two hemorrhagic cases were both female, and occurred within 24 h of postembolization. The thromboembolic complication rates were not significantly different between the two groups. Oral administration of routine antiplatelet drugs for 3 days before stent-assisted coil embolization possibly increases the risk of delayed intracranial hemorrhage, compared to loading group. Symptomatic thromboembolic complications have no significant difference in the two different regimens.