Vascular dementia (VaD) is the most common cause of dementia in older adults. Due to the lack of effective treatment options, there is an urgent need to find an effective pharmaceutical compound to combat VaD. Piracetam has been reported to improve impaired cognitive function in a variety of conditions in both human and animal models. However, the role and mechanism of Piracetam in VaD remain unclear. Therefore this study aimed to elucidate the effect of Piracetam on a cellular model of VaD in vitro. We found that Piracetam enhanced the growth of OGD-stimulated SH-SY5Y cells. In addition, Piracetam inhibited the oxidative stress of OGD-stimulated SH-SY5Y cells. Further, Piracetam improved mitochondrial function of OGD-stimulated SH-SY5Y cells. Mechanistically, Piracetam inhibited the PI3K/Akt/mTOR pathway in OGD-stimulated SH-SY5Y cells. Collectively, Piracetam improved oxidative stress and mitochondrial dysfunction of OGD-stimulated SH-SY5Y cells through PI3K/Akt/mTOR axis. Hence, Piracetam has the potential to serve as a promising drug of VaD.

UNASSIGNED: Brain microvascular endothelial cell (BMEC) functions loss is a key event in the development of ischemic stroke, which may be affected by the dysregulation of circular RNAs (circRNAs). We aimed to unveil the role of circRNA FKBP Prolyl Isomerase 3 (circFKBP3) in cell models of ischemic stroke.
UNASSIGNED: Cell models of ischemic stroke were constructed in human BEMCs (HBMECs) with the treatment of oxygen glucose deprivation (OGD). Quantitative real-time PCR (qPCR) and western blotting were conducted for expression analysis of circFKBP3, miR-766-3p and TNF receptor associated factor 3 (TRAF3). CCK-8, transwell, wound healing, flow cytometry, tube formation and ELISA assays were implemented to monitor cell viability, migration, apoptosis, angiogenesis and inflammation production. The putative binding relationship between miR-766-3p and circFKBP3 or TRAF3 was validated by dual-luciferase, RIP and pull-down assays.
UNASSIGNED: CircFKBP3 expression was elevated in OGD-treated HBMECs. OGD suppressed HBMEC viability, migration, angiogenesis, and provoked cell apoptosis and inflammation production, while knockdown of circFKBP3 attenuated these effects. CircFKBP3 interacted with miR-766-3p, and circFKBP3 absence-repressed HBMEC function loss and inflammation were recovered by miR-766-3p inhibition. CircFKBP3 targeted miR-766-3p to regulate TRAF3 expression. MiR-766-3p enrichment-repressed HBMEC function loss and inflammation were recovered by TRAF3 overexpression.
UNASSIGNED: CircFKBP3 absence alleviated OGD-induced function loss and inflammatory responses of HBMECs via governing the miR-766-3p/TRAF3 axis.
CircFKBP3 expression is elevated in OGD-treated HBMECs.OGD-induced HBMEC function loss and inflammation are alleviated by circFKBP3 absence.CircFKBP3 directly targets miR-766-3p to regulate TRAF3 expression.

We used an in vitro model of the human brain immune microenvironment to simulate hypoxic-ischemic brain injury (HIBI) and treatment with human umbilical cord mesenchymal stem cells (hUMSCs) to address the transformation barriers of gene differences between animals and humans in preclinical research. A co-culture system, termed hNAME, consisted of human hippocampal neurons (N), astrocytes (A), microglia (M), and brain microvascular endothelial cells (E). Flow cytometry measured the apoptosis rates of neurons and endothelial cells. hNAME-neurons and endothelial cells experienced more severe damage than monolayer cells, particularly after 48 h and 24 h of reoxygenation (OGD48/R24). Western blotting identified neuroinflammatory response markers, including HIF-1α, C1q, C3, TNF-α, and iNOS. Inflammatory factors originated from the glial chamber rather than the neurons and vascular endothelial chambers. A gradual increase in the release of inflammatory factors was observed as the OGD and reoxygenation times increased, peaking at OGD48/R24. The hNAME value was confirmed in human umbilical cord mesenchymal stem cells (hUMSCs). Treatment with hUMSCs resulted in a notable decrease in the severity of neuronal and endothelial cell damage in hNAME. The hNAME is an ideal in vitro model for simulating the immune microenvironment of the human brain because of the interactions between neurons, vessels, astrocytes, and microglia.

Neuronal swelling after excitotoxic insults is implicated in neuronal injury and death in the developing brain, yet mitigating brain edema with osmotic and surgical interventions yields poor clinical outcomes. Importantly, neuronal swelling and its downstream consequences during early brain development remain poorly investigated. Using multiphoton Ca2+ imaging in vivo (P12-17) and in acute brain slices (P8-12), we explored Ca2+-dependent downstream effects after neuronal cytotoxic edema. We observed the translocation of cytosolic GCaMP6s into the nucleus of a subpopulation of neurons minutes after various excitotoxic insults. We used automated morphology-detection algorithms for neuronal segmentation and quantified the nuclear translocation of GCaMP6s as the ratio of nuclear and cytosolic intensity (N/C ratio). Elevated neuronal N/C ratios were correlated to higher Ca2+ loads and could occur independently of neuronal swelling. Electron microscopy revealed that the nuclear translocation was associated with increased nuclear pore size. Inhibiting calpains prevented elevated N/C ratios and neuronal swelling. Thus, our results indicate altered nuclear transport in a subpopulation of neurons shortly after injury in the developing brain, which can be used as an early biomarker of acute neuronal injury.

It is widely accepted that miRNAs play an important role in the pathogenesis of myocardial fibrosis. This study aimed to identify a new pathway of miR-212-5p in the activation of human cardiac fibroblasts (HCFs) induced by oxygen-glucose deprivation (OGD). First, we found that KLF4 protein was markedly decreased in OGD-induced HCFs. Then, bioinformatics analysis and verification experiments were used to identify the existence of an interaction of KLF4 with miR-212-5p. Functional experiments indicated that OGD significantly upregulated the expression of hypoxia inducible factor-1 alpha (HIF-1α) in HCFs, which positively regulated miR-212-5p transcription by binding to its promoter. MiR-212-5p inhibited the expression of Krüppel-like factor 4 (KLF4) protein by binding to the 3\' untranslated coding regions (UTRs) of KLF4 mRNA. Inhibition of miR-212-5p effectively inhibited the activation of OGD-induced HCFs by upregulating KLF4 expression and inhibited cardiac fibrosis in vivo and in vitro.

Reperfusion is the fundamental treatment for ischaemic stroke; however, many ischaemic stroke patients cannot undergo reperfusion treatment. Furthermore, reperfusion can cause ischaemic reperfusion injuries. This study aimed to determine the effects of reperfusion in an in vitro ischaemic stroke model-oxygen and glucose deprivation (OGD) (0.3% O2)-with rat pheochromocytoma (PC12) cells and cortical neurons. In PC12 cells, OGD resulted in a time-dependent increase in cytotoxicity and apoptosis, and reduction in MTT activity from 2 h onwards. Reperfusion following shorter periods (4 and 6 h) of OGD recovered apoptotic PC12 cells, whereas after 12 h, OGD increased LDH release. In primary neurons, 6 h OGD led to significant increase in cytotoxicity, reduction in MTT activity and dendritic MAP2 staining. Reperfusion following 6 h OGD increased the cytotoxicity. HIF-1a was stabilised by 4 and 6 h OGD in PC12 cells and 2 h OGD onwards in primary neurons. A panel of hypoxic genes were upregulated by the OGD treatments depending on the duration. In conclusion, the duration of OGD determines the mitochondrial activity, cell viability, HIF-1a stabilization, and hypoxic gene expression in both cell types. Reperfusion following OGD of short duration is neuroprotective, whereas OGD of long duration is cytotoxic.

Previous research has shown that cyclin-dependent kinases (Cdks) that play physiological roles in cell cycle regulation become activated in post-mitotic neurons after ischemic stroke, resulting in apoptotic neuronal death. In this article, we report our results using the widely used oxygen-glucose deprivation (OGD) in vitro model of ischemic stroke on primary mouse cortical neurons to investigate whether Cdk7, as part of the Cdk-activating kinase (CAK) complex that activates cell cycle Cdks, might be a regulator of ischemic neuronal death and may potentially constitute a therapeutic target for neuroprotection. We found no evidence of neuroprotection with either pharmacological or genetic invalidation of Cdk7. Despite the well-established idea that apoptosis contributes to cell death in the ischemic penumbra, we also found no evidence of apoptosis in the OGD model. This could explain the absence of neuroprotection following Cdk7 invalidation in this model. Neurons exposed to OGD seem predisposed to die in an NMDA receptor-dependent manner that could not be prevented further downstream. Given the direct exposure of neurons to anoxia or severe hypoxia, it is questionable how relevant OGD is for modeling the ischemic penumbra. Due to remaining uncertainties about cell death after OGD, caution is warranted when using this in vitro model to identify new stroke therapies.

Cerebral ischaemia‒reperfusion injury (IRI), during which neurons undergo oxygen-glucose deprivation/reoxygenation (OGD/R), is a notable pathological process in many neurological diseases. N1-methyladenosine (m1A) is an RNA modification that can affect gene expression and RNA stability. The m1A landscape and potential functions of m1A modification in neurons remain poorly understood. We explored RNA (mRNA, lncRNA, and circRNA) m1A modification in normal and OGD/R-treated mouse neurons and the effect of m1A on diverse RNAs. We investigated the m1A landscape in primary neurons, identified m1A-modified RNAs, and found that OGD/R increased the number of m1A RNAs. m1A modification might also affect the regulatory mechanisms of noncoding RNAs, e.g., lncRNA-RNA binding proteins (RBPs) interactions and circRNA translation. We showed that m1A modification mediates the circRNA/lncRNA‒miRNA-mRNA competing endogenous RNA (ceRNA) mechanism and that 3\' untranslated region (3\'UTR) modification of mRNAs can hinder miRNA-mRNA binding. Three modification patterns were identified, and genes with different patterns had intrinsic mechanisms with potential m1A-regulatory specificity. Systematic analysis of the m1A landscape in normal and OGD/R neurons lays a critical foundation for understanding RNA modification and provides new perspectives and a theoretical basis for treating and developing drugs for OGD/R pathology-related diseases.

BACKGROUND: Circular RNA (circRNA) has been found to play an important role in the progression of many diseases, including ischemic stroke. However, the regulatory mechanism of circSEC11A in ischemic stroke progression need to further investigation.
METHODS: Human brain microvascular endothelial cells (HBMECs) were stimulated by oxygen glucose deprivation (OGD). CircSEC11A, SEC11A mRNA and miR (microRNA)-29a-3p were quantified by quantitative real-time PCR (qRT-PCR). SEMA3A, BAX and BCL2 protein level was quantified by western blot. Oxidative stress, cell proliferation, angiogenesis and apoptosis abilities were gauged by oxidative stress assay kit, 5-Ethynyl-2\'-Deoxyuridine (EdU) staining, tube formation assay and flow cytometry assays, respectively. Direct relationship between miR-29a-3p and circSEC11A or SEMA3A was validated by dual-luciferase reporter assay, RIP assay and RNA pull-down assay.
RESULTS: CircSEC11A was upregulated in OGD-induced HBMECs. OGD promoted the oxidative stress and apoptosis and inhibited cell proliferation and angiogenesis, while circSEC11A knockdown relieved the effects. CircSEC11A functioned as the sponge for miR-29a-3p, and miR-29a-3p inhibitor reversed the effects of si-circSEC11A on OGD-induced HBMECs oxidative injuries. Moreover, SEMA3A served as the target gene of miR-29a-3p. MiR-29a-3p inhibition ameliorated OGD-induced HBMECs oxidative injuries, while SEMA3A overexpression rescued the impacts of miR-29a-3p mimic.
CONCLUSIONS: CircSEC11A promoted the malignant progression in OGD-induced HBMECs through the mediation of miR-29a-3p/SEMA3A axis. This study has provided the new insight into the underlying application of circSEC11A in cell model of ischemic stroke.

Carnosol is a phytopolyphenol (diterpene) found and extracted from plants of Mediterranean diet, which has anti-tumor, anti-inflammatory and antioxidant effects. However, its role in ischemic stroke has not been elucidated.
Primary neurons subjected to oxygen-glucose deprivation (OGD) was used to investigate the effect of carnosol in vitro. A mouse MCAO model was used to evaluate the effect of carnosol on ischemic stroke in vivo. The mRNA level of inflammatory and apoptosis-related genes was determined by RT-PCR. The protein level of total and phosphorylated AMPK was determined by WB. H&E and Immunofluorescent assay was used to investigate the necrosis, inflammation and apoptosis in brain tissue.
Carnosol protected the activity of primary neurons subjected to oxygen-glucose deprivation (OGD) in vitro, as well as inhibited inflammation and apoptosis. Furthermore, carnosol could significantly reduce the infarct and edema volume and protect against neurological deficit in vivo, and had a significant inhibitory effect on brain neuroinflammation and apoptosis. Mechanically, carnosol could activate AMPK, and the effect of carnosol on cerebral ischemia-reperfusion injury cell model could be abolished by AMPK phosphorylation inhibitor.
Carnosol has a protective effect on ischemic stroke, and this effect is achieved through AMPK activation. Our study demonstrates the protective effect of carnosol on cerebral ischemia-reperfusion injury and provides a new perspective for the clinical treatment of ischemic stroke.