Therapeutic Target In Cerebral Ischemia Research Articles

Panobinostat, a Histone Deacetylase Inhibitor, Reduces LPS-Induced Expression of Inducible Nitric Oxide Synthase in Rat Immortalized Microglia HAPI Cells.

Microglia, resident immune cells in the central nervous system (CNS), play a critical role in maintaining CNS homeostasis. However, microglia activated in response to brain injury produce various inflammatory mediators, including nitric oxide (NO) and proinflammatory cytokines, leading to considerable neuronal damage. NO generated by inducible NO synthase (iNOS) rapidly reacts with superoxide to form a highly toxic product, peroxynitrite. Therefore, iNOS is considered to be a putative therapeutic target for cerebral ischemia. Here, we examined the effects of panobinostat (Pano), a histone deacetylase inhibitor, on lipopolysaccharide (LPS)-induced iNOS expression using rat immortalized microglia HAPI cells. Pano inhibited LPS-induced expression of iNOS mRNA and NO production in a dose-dependent manner; however, it had little effect on the LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 or nuclear translocation of nuclear factor-κB (NF-κB). The interferon-β (IFN-β)/signal transducer and activator of transcription (STAT) pathway is essential for LPS-induced iNOS expression in macrophages/microglia. We also examined the effects of Pano on LPS-induced IFN-β signaling. Pano markedly inhibited LPS-induced IFN-β expression and subsequent tyrosine phosphorylation of STAT1. However, the addition of IFN-β restored the decreased STAT1 phosphorylation but not the decreased iNOS expression. In addition, Pano inhibited the LPS-increased expression of octamer binding protein-2 and interferon regulatory factor 9 responsible for iNOS expression, but IFN-β addition also failed to restore the decreased expression of these factors. Thus, we conclude that the inhibitory effects of Pano are due not only to the inhibition of the IFN-β/STAT axis but also to the downregulation of other factors not involved in this axis.

Increased serum SGLT2 and its potential diagnostic and prognostic value in patients with acute ischemic stroke

BackgroundRecently acquired data suggests that sodium-glucose cotransporter-2 (SGLT2) may be a therapeutic target for cerebral ischemia. The specific impact of SGLT2 in acute ischemic stroke (AIS) remains unknown. We aimed to explore the levels of SGLT2 in AIS patients and its association with functional prognosis. MethodsIn this study, 132 AIS patients and 44 healthy controls were recruited prospectively to determine serum SGLT2 levels. Logistic regression analysis was employed to assess the association between serum SGLT2 level and stroke risk as well as 3-month outcome. Receiver operating characteristic (ROC) curves were utilized to evaluate predictive values for blood biomarkers. ResultsSerum SGLT2 levels were significantly higher (P =.000) in AIS patients (47.1 (interquartile range [IQR]: 42.4–50.9) ng/mL) than healthy controls (35.7 (IQR: 28.6–39.5) ng/mL). The optimal SGLT2 cutoff point for diagnosing AIS was 39.55 ng/mL, with a sensitivity of 90.2 % and specificity of 77.3 %. Serum levels of SGLT2 were negatively correlated with the onset time of AIS (linear fit R2 = 0.056, P =.006), but were not associated with National Institutes of Health Stroke Scale (NIHSS) scores (r = 0.007, P >.05) and lesion volume (r = -0.151, P >.05). SGLT2 was not remarkably different between patients with unfavorable and favorable outcomes (46.7 (IQR: 41.9–49.6) ng/mL vs 47.6 (IQR: 42.5–51.9) ng/mL; P =.321). ConclusionsThe serum SGLT2 concentration may be a potential biomarker for the diagnosis of AIS. However, it does not exhibit any association with disease severity or functional prognosis.

Paeoniae Radix Rubra extract attenuates cerebral ischemia injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway

Ethnopharmacological relevancePaeoniae Radix Rubra (PRR), the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, has been widely used to promote blood circulation and eliminate blood stasis in Chinese clinical practice, but its effect on cerebral ischemia is still rarely reported. Aim of the studyThe present study aimed to assess the potential therapeutic possibilities of the extract of PRR (PRRE) on cerebral ischemia, further exploring the underlying mechanism, and preliminary screening of the corresponding active components. Materials and methodsThe neuroprotective effects of PRRE in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO) injury and mouse hippocampal neuronal cells (HT22 cell line) following oxidative stress were confirmed. The mechanism was investigated using immunohistochemical staining, western blotting, transmission electron microscopy (TEM), and immunofluorescence. The active components of PRRE were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and molecular docking. ResultsThe in vivo study showed that PRRE reduced infarct volume and improved neurological deficits in rats, and the expression of GPX4, FTH1, Beclin1, LC3 II, and p-Akt was upregulated in the rat hippocampi. In addition, the vitro research indicated that PRRE can also alleviate H2O2-induced HT22 cell damage by regulating cytokines such as malondialdehyde (MDA), reduced glutathione (GSH) and reactive oxygen species (ROS), and the expressions of GPX4 and Beclin1 were observed to be elevated. The PI3K/Akt signalling pathway was inhibited by LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K). Furthermore, the effective components of PRRE in regulating ferroptosis and autophagy are mainly defined as albiflorin, paeoniflorin, benzoyl paeoniflorin, oleanolic acid, and hederagenin. ConclusionPRRE exerts neuroprotective effects against cerebral ischaemic injury by inhibiting ferroptosis and activating autophagy through the PI3K/Akt signalling pathway. This study provides an experimental basis for the potential application of PRRE as a novel therapeutic drug, and PI3K/Akt-associated ferroptosis and autophagy as therapeutic targets for cerebral ischemia.

Mitochondrial pyruvate carrier 1 alleviates hypoxic-ischemic brain injury in rats

AimsMitochondrial dysfunction is a critical pathological change in cerebral ischemia. Mitochondrial pyruvate carrier 1 (MPC1) is a mitochondrial inner membrane protein carrier participating in pyruvate transport. The work is aiming to figure out the effect of MPC1 on cerebral ischemia. Main methodsBilateral internal carotid artery embolization (BICAO) model and oxygen glucose deprivation/reoxygenation (OGD/R) model were used to simulate cerebral ischemia in vivo and in vitro. The effect of MPC1 on cerebral ischemia was detected by imaging, behavioral test, immunofluorescence, flow cytometry, transmission electron microscopy, Western blot and RT-Q-PCR. RNA-sequence (RNA-seq) was applied to explore the potential molecular mechanisms underlying the role of MPC1 in cerebral ischemia. Key findingAfter BICAO or OGD/R treatment, MPC1 expression in ischemic cortical neurons was significantly decreased. And MPC1 deficiency significantly reduced cerebral blood flow, decreased locomotion activities and exacerbated neuronal injury. Moreover, MPC1 deficiency obviously aggravated oxidative stress, structural disruption and dysfunction of mitochondria, autophagy and calcium overload of ischemic cortical neurons. Interestingly, MPC1 overexpression remarkably reversed neuronal loss and persisting neuronal deficits induced by OGD. Using RNA-seq, 38 MPC1-associated differentially expressed genes were involved in oxidative stress, autophagy and calcium overload. Our results further confirmed that MPC1 could alleviate autophagy via the PI3K/Akt/mTOR pathway in the ischemic cortical neurons. SignificanceMPC1 may exert neuroprotective effects by attenuating oxidative stress, mitochondrial dysfunction, calcium overload and autophagy during cerebral ischemia. MPC1-related genes identified by RNA-seq may be a novel therapeutic target for cerebral ischemia.

Lactate attenuates astrocytic inflammation by inhibiting ubiquitination and degradation of NDRG2 under oxygen–glucose deprivation conditions

BackgroundBrain lactate concentrations are enhanced in response to cerebral ischemia and promote the formation of reactive astrocytes, which are major components of the neuroinflammatory response and functional recovery, following cerebral ischemia. NDRG2 is upregulated during reactive astrocyte formation. However, its regulation and function are unclear. We studied the relationship between lactate and NDRG2 in astrocytes under conditions of ischemia or oxygen–glucose deprivation (OGD).MethodsWe examined astrocytic NDRG2 expression after middle cerebral artery occlusion (MCAO) using western blot and immunofluorescence staining. Under hypoxia conditions, we added exogenous L-lactate sodium (lactate) to cultured primary astrocytes to explore the effects of lactate on the ubiquitination modification of NDRG2. We profiled the transcriptomic features of NDRG2 silencing in astrocytes after 8 h of OGD conditions as well as exogenous lactate treatment by performing RNA-seq. Finally, we evaluated the molecular mechanisms of NDRG2 in regulating TNFα under OGD conditions using western blot and immunohistochemistry.ResultsReactive astrocytes strongly expressed NDRG2 in a rat model of MCAO. We also showed that lactate stabilizes astrocytic NDRG2 by inhibiting its ubiquitination. NDRG2 inhibition in astrocytes increased inflammation and upregulated immune-associated genes and signaling pathways. NDRG2 knockdown induced TNFα expression and secretion via c-Jun phosphorylation.ConclusionsWe revealed that under OGD conditions, lactate plays an important anti-inflammatory role and inhibits TNFα expression by stabilizing NDRG2, which is beneficial for neurological functional recovery. NDRG2 may be a new therapeutic target for cerebral ischemia.

Stress conditions induced circRNAs profile of extracellular vesicles in brain microvascular endothelial cells.

Cerebral ischemia causes hypoxic injury and inflammation, and brain microvascular endothelial cells (BMVECs) dysfunction is an initial stage of blood-brain barrier disruption. Endothelial cells secrete extracellular vesicles (EVs) that are involved in intercellular signal transduction. EVs contain a variety of RNAs, proteins, and metabolites. Circular RNA (circRNA) is a member of the non-coding RNA. The expression profile and potential function of circRNAs in BMVECs are unknown. Here, human BMVECs have undergone hypoxia or TNF-α induction, and the changes in circRNAs were measured by RNA sequencing. A total of 70 circRNAs showed differential expression, including 43 previously unrecorded circRNAs and 27 recorded circRNAs. Since astrocyte end-feet encircle endothelial cells, they are considered the main targets of the EVs from BMVEC. The miRNA sequence data and bioinformatics were used to predict the circRNA-miRNA-mRNA networks in astrocytes. The gene ontology (GO) analysis showed the main downstream targets of circRNAs are DNA transcription regulation and protein kinase-related signaling pathways. These results suggest that altering circRNAs may be a potential therapeutic target for cerebral ischemia induced hypoxic injury and inflammation.

Downregulation of mitochondrial pyruvate carrier 2 aggravates neuronal injury in the cortex following cerebral ischemia in rat

Cerebral ischemia is one of the most common disabling and lethal diseases worldwide, but its underlying mechanisms remain unclear. Mitochondrial pyruvate carrier 2 (MPC2), a subunit of MPC complex, plays pivotal roles in coordinating glycolytic and mitochondrial activities. In the present study, the expression of MPC2 was significantly reduced in the ischemic cerebral cortex of rats at 24 h after bilateral internal carotid artery occlusion (BICAO), and in the cortical neurons after 1 h oxygen-glucose deprivation (OGD)/24 h reoxygenation treatment. After MPC2 gene knockdown, the number and expression of neurons were remarkably decreased in the ischemic cerebral cortex of BICAO rats and OGD-treated neurons. UK5099 significantly reduced the number, expression and viability of OGD-treated neurons, and resulted in a significant decrease in length of neurite. Using RNA-sequencing (RNA-seq) technique, we further identified MPC2-related differential genes in the ischemic cerebral cortex of BICAO rats. In conclusion, our results suggested that the decrease in MPC2 expression aggravated ischemic injury, and MPC2-related genes might be a novel therapeutic target for cerebral ischemia.

Nrf2 protects against cerebral ischemia-reperfusion injury by suppressing programmed necrosis and inflammatory signaling pathways.

BackgroundThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) -mediated neuroinflammation is linked to neuronal necroptosis in cerebral ischemia-reperfusion (I/R) injury, especially in cerebral ischemic penumbra. This study was designed to investigate the regulation of nuclear factor E2-related factor-2 (Nrf2) on NLRP3 inflammasome in necroptosis signal pathway induced by I/R injury.MethodsWe investigated the mechanisms of Nrf2-negative regulation in necroptosis signaling pathway by using middle cerebral artery occlusion (MCAO) with Q-VD-OPH injected intraperitoneally. The protein level of the NLRP3 inflammasome was detected by western blot with Nrf2 knockdown and overexpression. NLRP3 inflammasome activation was Reactive oxygen species (ROS) dependent, and the protein level was regulated when N-acetylcysteine (NAC) and adenosine triphosphate (ATP) were selected as tools for regulating ROS.ResultsWe demonstrated the negative regulation of Nrf2 on NLRP3 inflammasome activation in Q-VD-OPH-induced necroptosis in cerebral artery I/R injury through Lentivirus-mediated RNA Interferenc, which mediated knockdown and overexpression of Nrf2. NLRP3 inflammasome activation was sensitive to both ATP-mediated ROS level increases and NAC-mediated ROS inhibition, suggesting that ROS is associated with the activation of NLRP3 inflammasome in necroptosis. In addition, Nrf2-induced NAD(P)H quinone dehydrogenase 1 (NQO1) was involved in the inhibition of NLRP3 inflammasome activation. These results suggest that Nrf2 regulates NQO1 to attenuate ROS, which negatively regulates NLRP3 inflammasome.ConclusionsNrf2/NQO1 was an inhibitor of ROS-induced NLRP3 inflammasome activation in Q-VD-OPH-induced necroptosis following cerebral I/R injury. Therefore, NLRP3 inflammasome could be a potential therapeutic target for cerebral ischemia.

Fatty Acid-Binding Proteins Aggravate Cerebral Ischemia-Reperfusion Injury in Mice.

Fatty acid-binding proteins (FABPs) regulate the intracellular dynamics of fatty acids, mediate lipid metabolism and participate in signaling processes. However, the therapeutic efficacy of targeting FABPs as novel therapeutic targets for cerebral ischemia is not well established. Previously, we synthesized a novel FABP inhibitor, i.e., FABP ligand 6 [4-(2-(5-(2-chlorophenyl)-1-(4-isopropylphenyl)-1H-pyrazol-3-yl)-4-fluorophenoxy)butanoic acid] (referred to here as MF6). In this study, we analyzed the ability of MF6 to ameliorate transient middle cerebral artery occlusion (tMCAO) and reperfusion-induced injury in mice. A single MF6 administration (3.0 mg/kg, per os) at 0.5 h post-reperfusion effectively reduced brain infarct volumes and neurological deficits. The protein-expression levels of FABP3, FABP5 and FABP7 in the brain gradually increased after tMCAO. Importantly, MF6 significantly suppressed infarct volumes and the elevation of FABP-expression levels at 12 h post-reperfusion. MF6 also inhibited the promotor activity of FABP5 in human neuroblastoma cells (SH-SY5Y). These data suggest that FABPs elevated infarct volumes after ischemic stroke and that inhibiting FABPs ameliorated the ischemic injury. Moreover, MF6 suppressed the inflammation-associated prostaglandin E2 levels through microsomal prostaglandin E synthase-1 expression in the ischemic hemispheres. Taken together, the results imply that the FABP inhibitor MF6 can potentially serve as a neuroprotective therapeutic for ischemic stroke.

Activation of liver X receptor promotes hippocampal neurogenesis and improves long-term cognitive function recovery in acute cerebral ischemia-reperfusion mice.

Cerebral ischemia (CI) leads to cognitive dysfunction due to the loss of hippocampal neurons. Liver X receptors (LXRs), including the LXRα and LXRβ isoforms, are critical for neurogenesis, synaptic plasticity, neurodegeneration, and cholesterol metabolism. However, the potential role of LXRs in the pathogenesis of CI-induced cognitive impairment is unclear. Therefore, we investigated the effects of LXR activation on hippocampal neurogenesis and cognitive function in mice with CI. C57 mice were randomized into four groups that included a sham group and three treatment groups with CI [Vehicle, TO901317 (TO90, an agonist of LXRs) and GSK2033 (an antagonist of LXRs)]. Mice were subjected to bilateral common carotid artery occlusion for 20min to induce transient CI. The Morris water maze test was executed to detect spatial learning and memory. Proliferation, differentiation, and immature neurons in the subgranular zone (SGZ) were examined using Immunofluorescence. Western blot assay was used to detect the expression of the Wnt/β-catenin signaling pathway-associated protein. TO90 significantly improved spatial learning and memory deficits induced by CI on 28days. It enhanced the proliferation of neural stem cells, the number of immature neurons and the differentiation from nascent cells to neurons. The expression of the Wnt/β-catenin signaling pathway-associated protein level was totally increased. The forenamed effects of TO90 were decreased in GSK2033 group. Thus, our findings suggest that LXRs activation can improve long-term cognitive dysfunction caused by CI by increasing neurogenesis, and LXRs may serve as a potential therapeutic target for cerebral ischemia. Cover Image for this issue: doi: 10.1111/jnc.14753.

MiRNA-20b inhibits cerebral ischemia-induced inflammation through targeting NLRP3.

The present study was designed to investigate the role of microRNA (miRNA)-20b in the inflammatory response during cerebral ischemia and the underlying mechanism following cerebral ischemia. A reverse transcription quantitative polymerase chain reaction assay was used to measure the expression of miRNA-20b, and tumor necrosis factor α, interleukin (IL)-6, IL-18 and IL-1β levels were measured using ELISA. In addition, the protein expression levels of NOD-like receptor pyrin domain containing 3 (NLRP3), caspase-1, IL-1β and IL-18 were determined by western blot analysis. It was determined that the expression of miRNA-20b during cerebral ischemia was increased compared with the control group. The overexpression of miRNA-20b increased the levels of IL-1β and IL-18 in the cerebral ischemia group through activation of the NLRP3 signaling pathway. Conversely, the downregulation of miRNA-20b suppressed IL-1β and IL-18 levels in cerebral ischemia via suppression of the NLRP3 signaling pathway. Additionally, the overexpression of miRNA-20b increased the levels of adenosine 5′-triphosphate (ATP) and reactive oxygen species (ROS) in the cerebral ischemia group, which were decreased following the downregulation of miRNA-20b. The inhibition of NLRP3 decreased the pro-inflammatory effects of miRNA-20b in cerebral ischemia. Suppression of ATP decreases the pro-inflammatory effects of miRNA-20b in cerebral ischemia. Suppression of ROS also decreases the pro-inflammatory effects of miRNA-20b in cerebral ischemia. Collectively, the present study provided novel insight into the role of miRNA-20b upregulation in the promotion of inflammation following cerebral infarction, suggesting that the miRNA-20b/NLRP3 axis may be a putative therapeutic target in cerebral ischemia.

Thioredoxin as a Therapeutic Target in Cerebral Ischemia.

Cerebral ischemia is a common cause of disability and death. Ischemic brain injury results from complex pathological processes, including oxidative stress, inflammation, and apoptosis. Thioredoxin( Trx) is an important multifunctional protein, which regulates cellular redox status. Increasing studies have demonstrated that Trx provides a neuroprotective role against cerebral ischemia-induced injury. A systematic search of PMC and the PubMed Database was conducted to summarize the protective effects of Trx against cerebral ischemia. This article reviews the understanding of potential effects and mechanisms of Trx against cerebral ischemia, including the anti-oxidant, anti-apoptotic and anti-inflammatory effects, as well as the activation of prosurvival pathway. We also summarize that some natural compounds induce the expression of Trx, which is involved in their anti-ischemic effects. In conclusion, Trx has a potential neuroprotection in cerebral ischemia and may be very promising for clinical therapy of ischemic stroke in the future.

Sphingosine 1-phosphate receptor subtype 3 (S1P3) contributes to brain injury after transient focal cerebral ischemia via modulating microglial activation and their M1 polarization

BackgroundThe pathogenic roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling in cerebral ischemia have been evidenced mainly through the efficacy of FTY720 that binds non-selectively to four of the five S1P receptors (S1P1,3,4,5). Recently, S1P1 and S1P2 were identified as specific receptor subtypes that contribute to brain injury in cerebral ischemia; however, the possible involvement of other S1P receptors remains unknown. S1P3 can be the candidate because of its upregulation in the ischemic brain, which was addressed in this study, along with underlying pathogenic mechanisms.MethodsWe used transient middle cerebral artery occlusion/reperfusion (tMCAO), a mouse model of transient focal cerebral ischemia. To identify S1P3 as a pathogenic factor in cerebral ischemia, we employed a specific S1P3 antagonist, CAY10444. Brain damages were assessed by brain infarction, neurological score, and neurodegeneration. Histological assessment was carried out to determine microglial activation, morphological transformation, and proliferation. M1/M2 polarization and relevant signaling pathways were determined by biochemical and immunohistochemical analysis.ResultsInhibiting S1P3 immediately after reperfusion with CAY10444 significantly reduced tMCAO-induced brain infarction, neurological deficit, and neurodegeneration. When S1P3 activity was inhibited, the number of activated microglia was markedly decreased in both the periischemic and ischemic core regions in the ischemic brain 1 and 3 days following tMCAO. Moreover, inhibiting S1P3 significantly restored the microglial shape from amoeboid to ramified microglia in the ischemic core region 3 days after tMCAO, and it attenuated microglial proliferation in the ischemic brain. In addition to these changes, S1P3 signaling influenced the proinflammatory M1 polarization, but not M2. The S1P3-dependent regulation of M1 polarization was clearly shown in activated microglia, which was affirmed by determining the in vivo activation of microglial NF-κB signaling that is responsible for M1 and in vitro expression levels of proinflammatory cytokines in activated microglia. As downstream effector pathways in an ischemic brain, S1P3 influenced phosphorylation of ERK1/2, p38 MAPK, and Akt.ConclusionsThis study identified S1P3 as a pathogenic mediator in an ischemic brain along with underlying mechanisms, involving its modulation of microglial activation and M1 polarization, further suggesting that S1P3 can be a therapeutic target for cerebral ischemia.

Potential of the Cerebral Sodium-Glucose Transporter as a Novel Therapeutic Target in Cerebral Ischemia

Cerebral ischemic stress often induces a hyperglycemic condition. This postischemic hyperglycemia exacerbates the development of cerebral ischemic neuronal damage, although the mechanism of this exacerbation remains to be clarified. We previously discovered that the cerebral sodium-glucose transporter (SGLT) was closely involved in the development of cerebral ischemic neuronal damage. SGLT is a member of the glucose transporter family and moves glucose together with sodium ions. SGLT-1, -3, -4, and -6 are distributed in the brain. We conducted further experiments to elucidate the detailed mechanism of the exacerbation of cerebral ischemia by cerebral SGLT. The results clarified: 1) the relationship between cerebral SGLT and postischemic hyperglycemia; 2) the involvement of cerebral SGLT-1 (a cerebral SGLT isoform) in cerebral ischemic neuronal damage; and 3) the effects of sodium influx through cerebral SGLT on the development of cerebral ischemic neuronal damage. This paper presents our data on the involvement of cerebral SGLT in the exacerbation of cerebral ischemic neuronal damage.

Downregulation of microRNA-195 promotes angiogenesis induced by cerebral infarction via targeting VEGFA.

Angiogenesis, the formation of new blood vessels from preexisting endothelium, is a process that involves a series of interassociated and mutually interactive pathophysiological processes. It is accepted that microRNAs (miRNAs) regulate endothelial cell behavior, including their involvement in angiogenesis. However, it remains unclear whether miRNAs are involved in the regulation of angiogenesis following cerebral ischemia. Therefore, the present study aimed to investigate the role of miRNAs in angiogenesis and the underlying mechanism following cerebral ischemia. Expression profiles of miRNAs in rat brain samples following middle cerebral artery occlusion (MCAO) were investigated using a miRNA microarray. The expression of candidate miRNA, miR-195 was further validated using reverse transcription-quantitative polymerase chain reaction. Then, the effects of miR-195 on cell migration and tube formation of human umbilical vein vascular endothelial cells (HUVECs) were investigated following miR-195 silencing, and overexpression. The specific target genes of miR-195 were predicted using microRNA prediction bioinformatics software (http://www.microrna.org/microrna/home.do), and then confirmed using a dual-luciferase reporter assay and rescue experiment. It was demonstrated that miR-195 was significantly downregulated in the brains of rats following MCAO and in hypoxia-induced HUVECs. Furthermore, it was revealed that miR-195 overexpression inhibited the invasion ability and tube formation of HUVECs in vitro, while miR-195 silencing enhanced these functions. In addition, vascular endothelial growth factor A (VEGFA) was identified as a direct target of miR-195 and was negatively correlated with miR-195 expression. In addition, the rescue experiment revealed that overexpression of VEGFA reversed the inhibitory effects of miR-195 overexpression on the invasion ability and tube formation of HUVECs. The present study has provided a novel insight into the promoting roles of miR-195 downregulation on angiogenesis following cerebral infarction and suggests that the miR-195/VEGFA signaling pathway is a putative therapeutic target in cerebral ischemia.

The NAD+-Dependent Family of Sirtuins in Cerebral Ischemia and Preconditioning.

Sirtuins are an evolutionarily conserved family of NAD+-dependent lysine deacylases and ADP ribosylases. Their requirement for NAD+ as a cosubstrate allows them to act as metabolic sensors that couple changes in the energy status of the cell to changes in cellular physiological processes. NAD+ levels are affected by several NAD+-producing and NAD+-consuming pathways as well as by cellular respiration. Thus their intracellular levels are highly dynamic and are misregulated in a spectrum of metabolic disorders including cerebral ischemia. This, in turn, compromises several NAD+-dependent processes that may ultimately lead to cell death. Recent Advances: A number of efforts have been made to replenish NAD+ in cerebral ischemic injuries as well as to understand the functions of one its important mediators, the sirtuin family of proteins through the use of pharmacological modulators or genetic manipulation approaches either before or after the insult. Critical Issues and Future Directions: The results of these studies have regarded the sirtuins as promising therapeutic targets for cerebral ischemia. Yet, additional efforts are needed to understand the role of some of the less characterized members and to address the sex-specific effects observed with some members. Sirtuins also exhibit cell-type-specific expression in the brain as well as distinct subcellular and regional localizations. As such, they are involved in diverse and sometimes opposing cellular processes that can either promote neuroprotection or further contribute to the injury; which also stresses the need for the development and use of sirtuin-specific pharmacological modulators. Antioxid. Redox Signal. 28, 691-710.

Abstract WP293: Necroptosis Cell Death Signaling Amplifies Acute Hyperglycemic Stroke Injury

Introduction: Acidic pH is an important feature of the microenvironment of cerebral ischemic injury. Acute hyperglycemia has been used as a tool to augment intraischemic acidosis, oxidative stress, edema, and cell necrosis. In addition to classical unregulated necrosis, neurons can undergo regulated necrosis. One form of regulated cell death is RIP1-regulated necroptosis. However, the effect of acidosis on the RIP1 signaling pathway has not been well studied. Hypothesis: The acidic microenvironment associated with hyperglycemic middle cerebral artery occlusion (MCAO) amplifies oxidative stress, which then augments RIP1-regulated necroptosis. Methods: MCAO was produced for 1 h in male Sv/Ev129 mice by the filament technique. To produce acute hyperglycemia, mice received an ip infusion of 25% dextrose before and during MCAO to increase blood glucose into the 350-400 mg/dL range and further decrease ischemic tissue pH from 6.8 to 6.4. Results: As expected, hyperglycemic mice had a greater infarct volume (41.4±4.9% of hemisphere) than did normoglycemic mice (28.4±5.3%). With intraventricular injection of the RIP-1 kinase inhibitor necrostatin-1, infarct volume decreased significantly in hyperglycemic mice (14.9±3.1%). Hyperglycemic MCAO increased protein carbonyls and 3-nitrotyrosine to a greater extent than normoglycemic MCAO, whereas necrostatin-1 attenuated their formation in hyperglycemic mice. Using immunoprecipitation, we found that hyperglycemic MCAO augmented the association of RIP3 with RIP1, MLKL, and PGAM5 and that these associations were blocked by necrostatin-1. Conclusions: These results indicate that RIP1-regulated necroptosis can make a major contribution to the ischemic neuronal injury that is augmented by acute hyperglycemia. The mechanism of augmentation of oxidative stress by intraischemic acidosis appears to require RIP1 kinase activity because necrostatin-1 attenuated protein carbonyl and 3-nitrotyrosine formation. Because much of the cell death appears to result from regulated necrosis rather than classical unregulated necrosis, RIP1-regulated necroptosis may serve as a therapeutic target for cerebral ischemia associated with severe acidosis.

β-Caryophyllene protects invitro neurovascular unit against oxygen-glucose deprivation and re-oxygenation-induced injury.

β-Caryophyllene (BCP) mediates neuroprotection in cerebral ischemic animals. The neurovascular unit (NVU) acts as an intricate network to maintain the neuronal homeostatic microenvironment. However, the effects exerted by BCP on NVU remain unclear. Therefore, we established an invitro NVU model to investigate the effects of BCP on oxygen-glucose deprivation and re-oxygenation (OGD/R)-induced injury. This model involved the co-culture of brain microvascular endothelial cells, neurons, and astrocytes. BCP (10μmol/L) was applied for 24h prior to OGD/R and maintained throughout OGD/R. Blood-brain barrier (BBB) integrity and neuronal apoptosis were analyzed. BCP pre-treatment prior to the initiation of OGD/R significantly (i) decreased BBB permeability and neuronal apoptosis, (ii) mitigated oxidative stress damage and the release of inflammatory cytokines, (iii) down-regulated Bax expression, metalloproteinase-9 activity and expression, and (iv) up-regulated claudin-5, occludin, ZO-1, growth-associated protein-43 and Bcl-2 expression. Thus, BCP pre-treatment exerted multiple protective effects on NVU in the context of OGD/R-induced injury. These protective effects potentially occur via reductions in oxidative stress damage and inflammatory cytokines that induce BBB breakdown, subsequently resulting in reduced neuronal apoptosis. The NVU serves as putative therapeutic targets for cerebral ischemia, and the results of this study provide new insights for the application of BCP as a neuroprotective agent.

Post-ischemic salubrinal treatment results in a neuroprotective role in global cerebral ischemia.

This study describes the neuroprotective effect of treatment with salubrinal 1 and 24h following 15min of ischemia in a two-vessel occlusion model of global cerebral ischemia. The purpose of this study was to determine if salubrinal, an enhancer of the unfolded protein response, reduces the neural damage modulating the inflammatory response. The study was performed in CA1 and CA3 hippocampal areas as well as in the cerebral cortex whose different vulnerability to ischemic damage is widely described. Characterization of proteins was made by western blot, immunofluorescence, and ELISA, whereas mRNA levels were measured by Quantitative PCR. The salubrinal treatment decreased the cell demise in CA1 at 7days as well as the levels of matrix metalloprotease 9 (MMP-9) in CA1 and cerebral cortex at 48h and ICAM-1 and VCAM-1 cell adhesion molecules. However, increases in tumor necrosis factor α and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inflammatory markers were observed at 24h. Glial fibrillary acidic protein levels were not modified by salubrinal treatment in CA1 and cerebral cortex. We describe a neuroprotective effect of the post-ischemic treatment with salubrinal, measured as a decrease both in CA1 cell demise and in the blood-brain barrier impairment. We hypothesize that the ability of salubrinal to counteract the CA1 cell demise is because of a reduced ability of this structure to elicit unfolded protein response which would account for its greater ischemic vulnerability. Data of both treated and non-treated animals suggest that the neurovascular unit present a structure-dependent response to ischemia and a different course time for CA1/cerebral cortex compared with CA3. Finally, our study reveals a high responsiveness of endothelial cells to salubrinal in contrast to the limited responsiveness of astrocytes. The alleviation of ER stress by enhancing UPR with salubrinal treatment reduces the ischemic damage. This effect varies across the different neurovascular unit cell types. The salubrinal neuroprotective effect on CA1 supports differences in neurovascular unit for different brain regions and involves the inflammatory response and its time course. Thus, UPR modulation could be a therapeutic target in cerebral ischemia.

Identification of New Therapeutic Targets for Cerebral Ischemia by Genome-Wide Analysis

Stroke is a devastating condition afflicting mostly the elderly for which no viable medication exists to improve neuro-rehabilitation. In particular, great clinical benefit may accrue from deciphering and targeting basic neurobiological mechanisms underlying post-stroke CNS recovery both in structural and functional terms.