Cystatin C loaded in brain-derived extracellular vesicles rescues synapses after ischemic insult in vitro and in vivo.

Synaptic loss is an early event in the penumbra area after an ischemic stroke. Promoting synaptic preservation in this area would likely improve functional neurological recovery. We aimed to detect proteins involved in endogenous protection mechanisms of synapses in the penumbra after stroke and to analyse potential beneficial effects of these candidates for a prospective stroke treatment. For this, we performed Liquid Chromatography coupled to Mass Spectrometry (LC-MS)-based proteomics of synaptosomes isolated from the ipsilateral hemispheres of mice subjected to experimental stroke at different time points (24 h, 4 and 7 days) and compared them to sham-operated mice. Proteomic analyses indicated that, among the differentially expressed proteins between the two groups, cystatin C (CysC) was significantly increased at 24 h and 4 days following stroke, before returning to steady-state levels at 7 days, thus indicating a potential transient and intrinsic rescue mechanism attempt of neurons. When CysC was applied to primary neuronal cultures subjected to an in vitro model of ischemic damage, this treatment significantly improved the preservation of synaptic structures. Notably, similar effects were observed when CysC was loaded into brain-derived extracellular vesicles (BDEVs). Finally, when CysC contained in BDEVs was administered intracerebroventricularly to stroked mice, it significantly increased the expression of synaptic markers such as SNAP25, Homer-1, and NCAM in the penumbra area compared to the group supplied with empty BDEVs. Thus, we show that CysC-loaded BDEVs promote synaptic protection after ischemic damage in vitro and in vivo, opening the possibility of a therapeutic use in stroke patients.

Melanocortin Derivatives Induced Vascularization and Neuroglial Proliferation in the Rat Brain under Conditions of Cerebral Ischemia.

Stroke remains the second leading cause of death worldwide. The development of new therapeutic agents focused on restoring vascular function and neuroprotection of viable tissues is required. In this study the neuroprotective activity of melanocortin-like ACTH(4-7)PGP and ACTH(6-9)PGP peptides was investigated in rat brain at 24 h after transient middle cerebral artery occlusion (tMCAO). The severity of ischemic damage, changes in the proliferative activity of neuroglial cells and vascularization of rat brain tissue were analyzed. The administration of peptides resulted in a significant increase in the volume density of neurons in the perifocal zone of infarction compared to rats subjected to ischemia and receiving saline. Immunohistochemical analysis of the proliferative activity of neuroglia cells using PCNA antibodies showed a significant increase in the number of proliferating cells in the penumbra and in the intact cerebral cortex of rats receiving peptide treatment. The effect of peptides on vascularization was examined using CD31 antibodies under tMCAO conditions, revealing a significant increase in the volume density of vessels and their sizes in the penumbra after administration of ACTH(4-7)PGP and ACTH(6-9)PGP. These findings confirm the neuroprotective effect of peptides due to the activation of neuroglia proliferation and the enhancement of collateral blood flow.

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

Circular RNA CircPDS5B impairs angiogenesis following ischemic stroke through its interaction with hnRNPL to inactivate VEGF-A.

BACKGROUND: Ischemic stroke (IS) is the primary cause of mortality and disability worldwide. Circular RNAs (circRNAs) have been proposed as crucial regulators in IS. This study focused on the role of circPDS5B in IS and its underlying mechanism. METHOD: Transient middle cerebral artery occlusion (tMCAO) mice and glucose deprivation/reoxygenation (OGD/R)-exposed human brain microvascular endothelial cells (BMECs) were used as IS models. Expression levels of circPDS5B, heterogenous nuclear ribonucleoprotein L (hnRNPL), runt-related transcription factor-1 (Runx1), and Zinc finger protein 24 (ZNF24) were quantified by qRT-PCR. MTT, wound healing, transwell and tube formation assays were employed to evaluate the cell proliferation, migration, and angiogenesis, respectively. Moreover, RNA pull-down, and RIP assay were performed to investigate the interaction among circPDS5B, hnRNPL and vascular endothelial growth factor-A (VEGF-A). RESULTS: circPDS5B was significantly up-regulated in IS patients and tMCAO mice. Deficiency of circPDS5B relieved brain infarction and neuronal injury of tMCAO mice. OGD/R-induced apoptosis, inhibition in viability, migration, and angiogenesis in BMECs were dramatically abrogated by circPDS5B knockdown. Mechanistically, circPDS5B stabilized Runx1 and ZNF24 via recruiting hnRNPL, thereby suppressing the transcription and expression of VEGFA. hnRNPL silencing strengthened circPDS5B knockdown-mediated beneficial effect on IS. CONCLUSION: Altogether, our study showed that high expression of circPDS5B exacerbated IS through recruitment of hnRNPL to stabilize Runx1/ZNF24 and subsequently inactivate VEGFA. Our findings suggest circPDS5B may be a novel therapeutic target for IS.

Dl-3-n-butylphthalide promotes angiogenesis in ischemic stroke mice through upregulating autocrine and paracrine sonic hedgehog.

Dl-3-n-butylphthalide (NBP) is a small-molecule drug used in the treatment of ischemic stroke in China, which is proven to ameliorate the symptoms of ischemic stroke and improve the prognosis of patients. Previous studies have shown that NBP accelerates recovery after stroke by promoting angiogenesis. In this study, we investigated the mechanisms underlying the angiogenesis-promoting effects of NBP in ischemic stroke models in vitro and in vivo. OGD/R model was established in human umbilical vein endothelial cells (HUVECs) and human brain microvascular endothelial cells (HBMECs), while the tMCAO model was established in mice. The cells were pretreated with NBP (10, 50, 100 µM); the mice were administered NBP (4, 8 mg/kg, i.v.) twice after tMCAO. We showed that NBP treatment significantly stimulated angiogenesis by inducing massive production of angiogenic growth factors VEGFA and CD31 in both in vitro and in vivo models of ischemic stroke. NBP also increased the tubule formation rate and migration capability of HUVECs in vitro. By conducting the weighted gene co-expression network analysis, we found that these effects were achieved by upregulating the expression of a hedgehog signaling pathway. We demonstrated that NBP treatment not only changed the levels of regulators of the hedgehog signaling pathway but also activated the transcription factor Gli1. The pro-angiogenesis effect of NBP was abolished when the hedgehog signaling pathway was inhibited by GDC-0449 in HUVECs, by Sonic Hedgehog(Shh) knockdown in HUVECs, or by intracerebroventricular injection of AAV-shRNA(shh)-CMV in tMCAO mice. Furthermore, we found that HUVECs produced a pro-angiogenic response not only to autocrine Shh, but also to paracrine Shh secreted by astrocytes. Together, we demonstrate that NBP promotes angiogenesis via upregulating the hedgehog signaling pathway. Our results provide an experimental basis for the clinical use of NBP.

Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content with potential relevance to inflammation and recovery processes.

Extracellular vesicles (EVs) are lipid bilayer-enclosed structures that represent newly discovered means for cell-to-cell communication as well as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA, eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 h after experimental stroke in mice and compared them to controls (shams) using nCounter® Nanostring panels, with or without prior RNA isolation. We found that both panels show similar results when comparing upregulated mRNAs in stroke. Notably, the highest upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, thus indicating that regenerative mechanisms already take place at this time-point. The five top overrepresented mRNAs in stroke mice were confirmed by RT-qPCR and, interestingly, found to be full-length. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and predominantly present in small BDEVs (≤ 200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation, and that the mRNA cargo of BDEVs indicates a possible participation in inflammatory but also recovery processes after stroke.

l-borneol promotes neurovascular unit protection in the subacute phase of transient middle cerebral artery occlusion rats: p38-MAPK pathway activation, anti-inflammatory, and anti-apoptotic effect.

Our previous study showed l-borneol reduced cerebral infarction in the acute stage after cerebral ischemia, but there is little about the study of subacute phase. We herein investigated the cerebral protective effects of l-borneol on neurovascular units (NVU) in the subacute phase after transient middle cerebral artery occlusion (t-MCAO). The t-MCAO model was prepared by the line embolus method. Zea Longa, mNss, HE, and TTC staining were used to evaluate the effect of l-borneol. We evaluated the mechanisms of l-borneol on inflammation, p38 MAPK pathway, and apoptosis, etc. through various technologies. l-borneol 0.2, 0.1, 0.05 g·kg-1 could significantly reduce cerebral infarction rate, alleviate the pathological injury, and inhibit inflammation reaction. l-borneol could also significantly increase brain blood supply, Nissl bodies, and the expression of GFAP. Additionally, l-borneol activated the p38 MAPK signaling pathway, inhibited cell apoptosis, and maintained BBB integrity. l-borneol had a neuroprotective effect, which was related to activating the p38 MAPK signaling pathway, inhibiting inflammatory response and apoptosis, and improving cerebral blood supply to protect BBB and stabilize and remodel NVU. The study will provide a reference for the use of l-borneol in the treatment of ischemic stroke in the subacute phase.

ERK1/2 Activity Is Critical for the Outcome of Ischemic Stroke.

Ischemic disorders are the leading cause of death worldwide. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are thought to affect the outcome of ischemic stroke. However, it is under debate whether activation or inhibition of ERK1/2 is beneficial. In this study, we report that the ubiquitous overexpression of wild-type ERK2 in mice (ERK2wt) is detrimental after transient occlusion of the middle cerebral artery (tMCAO), as it led to a massive increase in infarct volume and neurological deficits by increasing blood-brain barrier (BBB) leakiness, inflammation, and the number of apoptotic neurons. To compare ERK1/2 activation and inhibition side-by-side, we also used mice with ubiquitous overexpression of the Raf-kinase inhibitor protein (RKIPwt) and its phosphorylation-deficient mutant RKIPS153A, known inhibitors of the ERK1/2 signaling cascade. RKIPwt and RKIPS153A attenuated ischemia-induced damages, in particular via anti-inflammatory signaling. Taken together, our data suggest that stimulation of the Raf/MEK/ERK1/2-cascade is severely detrimental and its inhibition is rather protective. Thus, a tight control of the ERK1/2 signaling is essential for the outcome in response to ischemic stroke.

Comparative Use of Contralateral and Sham-Operated Controls Reveals Traces of a Bilateral Genetic Response in the Rat Brain after Focal Stroke.

Ischemic stroke is a multifactorial disease with a complex etiology and global consequences. Model animals are widely used in stroke studies. Various controls, either brain samples from sham-operated (SO) animals or symmetrically located brain samples from the opposite (contralateral) hemisphere (CH), are often used to analyze the processes in the damaged (ipsilateral) hemisphere (IH) after focal stroke. However, previously, it was shown that focal ischemia can lead to metabolic and transcriptomic changes not only in the IH but also in the CH. Here, using a transient middle cerebral artery occlusion (tMCAO) model and genome-wide RNA sequencing, we identified 1941 overlapping differentially expressed genes (DEGs) with a cutoff value >1.5 and Padj < 0.05 that reflected the general transcriptome response of IH subcortical cells at 24 h after tMCAO using both SO and CH controls. Concomitantly, 861 genes were differentially expressed in IH vs. SO, whereas they were not vs. the CH control. Furthermore, they were associated with apoptosis, the cell cycle, and neurotransmitter responses. In turn, we identified 221 DEGs in IH vs. CH, which were non-DEGs vs. the SO control. Moreover, they were predominantly associated with immune-related response. We believe that both sets of non-overlapping genes recorded transcriptome changes in IH cells associated with transhemispheric differences after focal cerebral ischemia. Thus, the specific response of the CH transcriptome should be considered when using it as a control in studies of target brain regions in diseases that induce a global bilateral genetic response, such as stroke.

Pre-exposure of voglibose exerts cerebroprotective effects through attenuating activation of the polyol pathway and inflammation.

Chronic hyperglycemia induces activation of the polyol-sorbitol pathway, which is a major contributor to microvascular complications like stroke. The current study was designed to elucidate the therapeutic role of α-glucose inhibitor in chronic hyperglycemia-induced impaired polyol pathway and associated micro-complications. Male albino-Wistar rats (200-250 g) were treated with voglibose 10 mg kg-1  day-1 /p.o. for 2 weeks before middle cerebral artery occlusion; 72 hr after surgery, neurological score was evaluated and blood was collected for the assessment of various serum biochemical parameters like CRP, CK-MB, LDH, lipid profile, and blood glucose levels. In the end, brain samples were excised for determination of brain infarct volume, brain hemisphere weight difference, Na+-K+ ATPase activity oxidative stress-related parameters, aldose reductase activity, and gene expression studies. Results from the present study indicate that pre-treatment with voglibose showed significant improvement in lipid parameters but did not impact glucose levels. Voglibose has shown a statistically significant (p < .05) reduction in neurological score and brain infarct volume, and the difference in brain hemisphere weight as compared to the disease control group. Voglibose significantly (p < .05) improve all biochemical parameters and reduced Na+-K+ ATPase and aldose reductase activity. Moreover, voglibose produced a significant reduction in oxidative stress and down-regulation of TNF-α and BCl-2 gene expression which reduces the risk of factors related to stroke. In conclusion, the pleiotropic effect of voglibose on cerebrovascular complications may be due to inhibition of aldose reductase or anti-inflammatory pathways.

High-Frequency rTMS Improves Cognitive Function by Regulating Synaptic Plasticity in Cerebral Ischemic Rats.

Poststroke cognitive impairment (PSCI) is one of the most severe sequelae of stroke and lacks effective treatment. Previous studies have shown that high-frequency repetitive transcranial magnetic stimulation (rTMS) may be a promising PSCI therapeutic approach, but the underlying mechanism is unclear. To uncover the effect of rTMS on PSCI, a transient middle cerebral artery occlusion (tMCAO) model was established. Modified Neurological Severity Score (mNSS) test and Morris Water Maze (MWM) test were performed to assess the neurological and cognitive function of rats. Furthermore, to explore the underlying mechanism, differentially expressed genes (DEGs) in the hippocampus of rats in the rTMS group and tMCAO group were compared using RNA sequencing. Then, bioinformatics analysis, including gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and protein-protein interaction (PPI) network analysis, was conducted to elaborate these DEGs. Our results indicated that high-frequency rTMS could significantly improve neurological and cognitive function, according to mNSS and MWM tests. We found 85 DEGs, including 71 upregulated genes and 14 downregulated genes, between the rTMS group and tMCAO group. The major functional category was related to chemical synaptic transmission modulation and several DEGs were significantly upregulated in processes related to synaptic plasticity, such as glutamatergic synapses. Calb2, Zic1, Kcnk9, and Grin3a were notable in PPI analysis. These results demonstrate that rTMS has a beneficial effect on PSCI, and its mechanism may be related to the regulation of synaptic plasticity and functional genes such as Calb2, Zic1, Kcnk9, and Grin3a in the hippocampus.

The Mitochondrial mitoNEET Ligand NL-1 Is Protective in a Murine Model of Transient Cerebral Ischemic Stroke.

PURPOSE: Therapeutic strategies to treat ischemic stroke are limited due to the heterogeneity of cerebral ischemic injury and the mechanisms that contribute to the cell death. Since oxidative stress is one of the primary mechanisms that cause brain injury post-stroke, we hypothesized that therapeutic targets that modulate mitochondrial function could protect against reperfusion-injury after cerebral ischemia, with the focus here on a mitochondrial protein, mitoNEET, that modulates cellular bioenergetics. METHOD: In this study, we evaluated the pharmacology of the mitoNEET ligand NL-1 in an in vivo therapeutic role for NL-1 in a C57Bl/6 murine model of ischemic stroke. RESULTS: NL-1 decreased hydrogen peroxide production with an IC50 of 5.95 µM in neuronal cells (N2A). The in vivo activity of NL-1 was evaluated in a murine 1 h transient middle cerebral artery occlusion (t-MCAO) model of ischemic stroke. We found that mice treated with NL-1 (10 mg/kg, i.p.) at time of reperfusion and allowed to recover for 24 h showed a 43% reduction in infarct volume and 68% reduction in edema compared to sham-injured mice. Additionally, we found that when NL-1 was administered 15 min post-t-MCAO, the ischemia volume was reduced by 41%, and stroke-associated edema by 63%. CONCLUSION: As support of our hypothesis, as expected, NL-1 failed to reduce stroke infarct in a permanent photothrombotic occlusion model of stroke. This report demonstrates the potential therapeutic benefits of using mitoNEET ligands like NL-1 as novel mitoceuticals for treating reperfusion-injury with cerebral stroke.

Transient Focal Cerebral Ischemia Leads to miRNA Alterations in Different Brain Regions, Blood Serum, Liver, and Spleen.

Ischemic stroke is characterized by an occlusion of a cerebral blood vessel resulting in neuronal cell death due to nutritional and oxygen deficiency. Additionally, post-ischemic cell death is augmented after reperfusion. These events are paralleled by dysregulated miRNA expression profiles in the peri-infarct area. Understanding the underlying molecular mechanism in the peri-infarct region is crucial for developing promising therapeutics. Utilizing a tMCAo (transient Middle Cerebral Artery occlusion) model in rats, we studied the expression levels of the miRNAs (miR) 223-3p, 155-5p, 3473, and 448-5p in the cortex, amygdala, thalamus, and hippocampus of both the ipsi- and contralateral hemispheres. Additionally, the levels in the blood serum, spleen, and liver and the expression of their target genes, namely, Nlrp3, Socs1, Socs3, and Vegfa, were assessed. We observed an increase in all miRNAs on the ipsilateral side of the cerebral cortex in a time-dependent manner and increased miRNAs levels (miR-223-3p, miR-3473, and miR-448-5p) in the contralateral hemisphere after 72 h. Besides the cerebral cortex, the amygdala presented increased expression levels, whereas the thalamus and hippocampus showed no alterations. Different levels of the investigated miRNAs were detected in blood serum, liver, and spleen. The gene targets were altered not only in the peri-infarct area of the cortex but selectively increased in the investigated non-affected brain regions along with the spleen and liver during the reperfusion time up to 72 h. Our results suggest a supra-regional influence of miRNAs following ischemic stroke, which should be studied to further identify whether miRNAs are transported or locally upregulated.

Treatment with Edoxaban Attenuates Acute Stroke Severity in Mice by Reducing Blood-Brain Barrier Damage and Inflammation.

Patients with atrial fibrillation and previous ischemic stroke (IS) are at increased risk of cerebrovascular events despite anticoagulation. In these patients, treatment with non-vitamin K oral anticoagulants (NOAC) such as edoxaban reduced the probability and severity of further IS without increasing the risk of major bleeding. However, the detailed protective mechanism of edoxaban has not yet been investigated in a model of ischemia/reperfusion injury. Therefore, in the current study we aimed to assess in a clinically relevant setting whether treatment with edoxaban attenuates stroke severity, and whether edoxaban has an impact on the local cerebral inflammatory response and blood-brain barrier (BBB) function after experimental IS in mice. Focal cerebral ischemia was induced by transient middle cerebral artery occlusion in male mice receiving edoxaban, phenprocoumon or vehicle. Infarct volumes, functional outcome and the occurrence of intracerebral hemorrhage were assessed. BBB damage and the extent of local inflammatory response were determined. Treatment with edoxaban significantly reduced infarct volumes and improved neurological outcome and BBB function on day 1 and attenuated brain tissue inflammation. In summary, our study provides evidence that edoxaban might exert its protective effect in human IS by modulating different key steps of IS pathophysiology, but further studies are warranted.

Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4-7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia-Reperfusion.

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

The receptor-receptor interaction between mGluR1 receptor and NMDA receptor: a potential therapeutic target for protection against ischemic stroke.

Ischemic stroke is one of the leading causes of long-term disability worldwide. It arises when the blood flow to the brain is severely impaired, causing brain infarction. The current therapies for ischemic stroke are tissue plasminogen activator and mechanical thrombectomy, which re-establishes blood circulation to the brain but offers no neuroprotective effects. Excitotoxicity, particularly through the N-methyl-d-aspartate receptor (NMDAR), has been heavily implicated in the pathophysiology of brain infarction resulting from ischemic stroke. Here we investigated the interaction between NMDAR and metabotropic glutamate receptor 1 (mGluR1) as a novel target to develop potential neuroprotective agents for ischemic stroke. Through coimmunoprecipitation and affinity binding assay, we revealed that the interaction is mediated through 2 distinct sites on the mGluR1 C terminus. We then found that the disruption of mGluR1-GluN2A subunit of NMDAR (GluN2A) protected the primary mouse hippocampal neurons against NMDAR-mediated excitotoxicity and reversed the NMDAR-mediated regulation of ERK1/2 in rat hippocampal slices. The same protection was also observed in an animal model of ischemic stroke, alleviating brain infarction and yielding better motor recovery. These findings confirmed the existence of a receptor-receptor interaction between NMDAR and mGluR1, implicating this interconnection as a potential treatment target site for ischemic stroke.-Lai, T. K. Y., Zhai, D. Su, P., Jiang, A., Boychuk, J., Liu, F. The receptor-receptor interaction between mGluR1 receptor and NMDA receptor: a potential therapeutic target for protection against ischemic stroke.

Ginsenoside Rg1 protects against ischemic/reperfusion-induced neuronal injury through miR-144/Nrf2/ARE pathway.

Ginsenoside Rg1 (Rg1), a saponin extracted from Panax ginseng, has been well documented to be effective against ischemic/reperfusion (I/R) neuronal injury. However, the underlying mechanisms remain obscure. In the present study, we investigated the roles of Nrf2 and miR-144 in the protective effects of Rg1 against I/R-induced neuronal injury. In OGD/R-treated PC12 cells, Rg1 (0.01-1 µmol/L) dose-dependently attenuated the cell injury accompanied by prolonging nuclear accumulation of Nrf2, enhancing the transcriptional activity of Nrf2, as well as promoting the expression of ARE-target genes. The activation of the Nrf2/ARE pathway by Rg1 was independent of disassociation with Keap1, but resulted from post-translational regulations. Knockdown of Nrf2 abolished all the protective changes of Rg1 in OGD/R-treated PC12 cells. Furthermore, Rg1 treatment significantly decreased the expression of miR-144, which downregulated Nrf2 production by targeting its 3'-untranlated region after OGD/R. Knockdown of Nrf2 had no effect on the expression of miR-144, suggesting that miR-144 was an upstream regulator of Nrf2. We revealed that there was a direct binding between Nrf2 and miR-144 in PC12 cells. Application of anti-miR-144 occluded the activation of the Nrf2/ARE pathway by Rg1 in OGD/R-treated PC12 cells. In tMCAO rats, administration of Rg1 (20 mg/kg) significantly alleviated ischemic injury, and activated Nrf2/ARE pathway. The protective effects of Rg1 were abolished by injecting of AAV-HIF-miR-144-shRNA into the predicted ischemic penumbra. In conclusion, our results demonstrate that Rg1 alleviates oxidative stress after I/R through inhibiting miR-144 activity and subsequently promoting the Nrf2/ARE pathway at the post-translational level.

Genome-wide transcriptome analysis using RNA-Seq reveals a large number of differentially expressed genes in a transient MCAO rat model.

BACKGROUND: The transient middle cerebral artery occlusion (tMCAO) model is used for studying the molecular mechanisms of ischemic damage and neuroprotection. Numerous studies have demonstrated the role of individual genes and associated signaling pathways in the pathogenesis of ischemic stroke. Here, the tMCAO model was used to investigate the genome-wide response of the transcriptome of rat brain tissues to the damaging effect of ischemia and subsequent reperfusion. RESULTS: Magnetic resonance imaging and histological examination showed that the model of focal ischemia based on endovascular occlusion of the right middle cerebral artery for 90 min using a monofilament, followed by restoration of the blood flow, led to reproducible localization of ischemic damage in the subcortical structures of the brain. High-throughput RNA sequencing (RNA-Seq) revealed the presence of differentially expressed genes (DEGs) in subcortical structures of rat brains resulting from hemisphere damage by ischemia after tMCAO, as well as in the corresponding parts of the brains of sham-operated animals. Real-time reverse transcription polymerase chain reaction expression analysis of 20 genes confirmed the RNA-Seq results. We identified 469 and 1939 genes that exhibited changes in expression of > 1.5-fold at 4.5 and 24 h after tMCAO, respectively. Interestingly, we found 2741 and 752 DEGs under ischemia-reperfusion and sham-operation conditions at 24 h vs. 4.5 h after tMCAO, respectively. The activation of a large number of genes involved in inflammatory, immune and stress responses, apoptosis, ribosome function, DNA replication and other processes was observed in ischemia-reperfusion conditions. Simultaneously, massive down-regulation of the mRNA levels of genes involved in the functioning of neurotransmitter systems was recorded. A Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that dozens of signaling pathways were associated with DEGs in ischemia-reperfusion conditions. CONCLUSIONS: The data obtained revealed a global profile of gene expression in the rat brain sub-cortex under tMCAO conditions that can be used to identify potential therapeutic targets in the development of new strategies for the prevention and treatment of ischemic stroke.

Neuroprotective Effects of Oxytocin Hormone after an Experimental Stroke Model and the Possible Role of Calpain-1.

BACKGROUND: Different mechanisms will be activated during ischemic stroke. Calpain proteases play a pivotal role in neuronal death after ischemia damage through apoptosis. Anti-apoptotic activities of the oxytocin (OT) in different ischemic tissues were reported in previous studies. Recently, a limited number of studies have noted the protective effects of OT in the brain. In the present study, the neuroprotective potential of OT in an animal model of transient middle cerebral artery occlusion (tMCAO) and the possible role of calpain-1 in the penumbra region were assessed. METHODS: Adult male Wistar rats underwent 1 hour of tMCAO and were treated with nasal administration of OT. After 24 hours of reperfusion, infarct size was evaluated by triphenyltetrazolium chloride. Immunohistochemical staining and Western blotting were used to examine the expression of calpain-1. Nissl staining was performed for brain tissue morphology evaluation. RESULTS: OT reduced the infarct volume of the cerebral cortex and striatum compared with the ischemia control group significantly (P < .05). Calpain-1 overexpression, which was caused by ischemia, decreased after OT administration (P < .05). The number of pyknotic nuclei in neurons increased dramatically in the ischemic area and OT attenuated the apoptosis of neurons in the penumbra region (P < .01). CONCLUSION: We provided evidence for the neuroprotective role of OT after tMCAO through calpain-1 attenuation.

[Effect of compatibility of ginkgolide A, ginkgolide B and ginkgolide K].

To investigate the best active compatibility of ginkgolide A, B and K (GA，GB，GK). The effects of GA, GB, GK alone, combinations of each two of them, and combinations of these three components on platelet-activating factor (PAF)-induced platelet aggregation activity and rat cerebral ischemia reperfusion model (tMCAO) were compared in this study. Different compatibilities of GA, GB and GK could significantly reduce the maximum aggregation rate of PAF-induced platelet aggregation, and the effect was most obvious in combination of the three. Different compatibilities of GA, GB and GK could alleviate the neural function, cerebral infarction volume and cerebral edema in the tMCAO model of rats to different degrees, and the effect of combinations of the three was stronger than those of combinations of two and single use. The combination of all of GA, GB and GK had the strongest effect on nerve injury caused by anti-platelet aggregation in tMCAO rats.