Cerebral Ischemic Preconditioning Research Articles

Transformation of A1/A2 Astrocytes Participates in Brain Ischemic Tolerance Induced by Cerebral Ischemic Preconditioning via Inhibiting NDRG2.

Cerebral ischemic preconditioning (CIP) has been shown to improve brain ischemic tolerance against subsequent lethal ischemia. Reactive astrocytes play important roles in cerebral ischemia-reperfusion. Recent studies have shown that reactive astrocytes can be polarized into neurotoxic A1 phenotype (C3d) and neuroprotective A2 phenotype (S100A10). However, their role in CIP remains unclear. Here, we focused on the role of N-myc downstream-regulated gene 2 (NDRG2) in regulating the transformation of A1/A2 astrocytes and promoting to brain ischemic tolerance induced by CIP. A Sprague Dawley rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used. Rats were divided into the following six groups: (1) sham group; (2) CIP group: left middle cerebral artery was blocked for 10min; (3) MCAO/R group: left middle cerebral artery was blocked for 90min; (4) CIP + MCAO/R group: CIP was performed 72h before MCAO/R; (5) AAV-NDRG2 + CIP + MCAO/R group: adeno-associated virus (AAV) carrying NDRG2 was administered 14 days before CIP + MCAO/R; (6) AAV-Ctrl + CIP + MCAO/R group: empty control group. The rats were subjected to neurological evaluation 24h after the above treatments, and then were sacrificed for 2, 3, 5-triphenyltetraolium chloride staining, thionin staining, immunofluorescence and western blot analysis. In CIP + MCAO/R group, the neurological deficit scores decreased, infarct volume reduced, and neuronal density increased compared with MCAO/R group. Notably, CIP significantly increased S100A10 expression and the number of S100A10+/GFAP+ cells, and also increased NDRG2 expression. MCAO/R significantly decreased S100A10 expression and the number of S100A10+/GFAP+ cells yet increased C3d expression and the number of C3d+/GFAP+ cells and NDRG2 expression, and these trends were reversed by CIP + MCAO/R. Furthermore, over-expression of NDRG2 before CIP + MCAO/R, the C3d expression and the number of C3d+/GFAP+ cells increased, while S100A10 expression and the number of S100A10+/GFAP+ cells decreased. Meanwhile, over-expression of NDRG2 blocked the CIP-induced brain ischemic tolerance. Taken together, these results suggest that CIP exerts neuroprotective effects against ischemic injury by suppressing A1 astrocyte polarization and promoting A2 astrocyte polarization via inhibiting NDRG2 expression.

STAT4-Mediated Klotho Up-Regulation Contributes to the Brain Ischemic Tolerance by Cerebral Ischemic Preconditioning via Inhibiting Neuronal Pyroptosis.

Our previous study has proved that the Klotho up-regulation participated in cerebral ischemic preconditioning (CIP)-induced brain ischemic tolerance. However, the exact neuroprotective mechanism of Klotho in CIP remains unclear. We explored the hypothesis that STAT4-mediated Klotho up-regulation contributes to the CIP-induced brain ischemic tolerance via inhibiting neuronal pyroptosis. Firstly, the expressions of pyroptosis-associated proteins (i.e., NLRP3, GSDMD, pro-caspase-1, and cleaved caspase-1) in hippocampal CA1 region were determined during the process of brain ischemic tolerance. We found the expression of pyroptosis-associated proteins was significantly up-regulated in the ischemic insult (II) group, and showed no significant changes in the CIP group. The expression level of each pyroptosis-associated proteins was lower in the CIP + II group than that in the II group. Inhibition of Klotho expression increased the expression of pyroptosis-associated proteins in the CIP + II group and blocked the CIP-induced brain ischemic tolerance. Injection of Klotho protein decreased the expression of pyroptosis-associated proteins in the II group, and protected neurons from ischemic injury. Secondly, the transcription factor STAT4 of Klotho was identified by bioinformatic analysis. Double luciferase reporter gene assay and chromatin immunoprecipitation assay showed STAT4 can bind to the site between nt - 881 and - 868 on the Klotho promoter region and positively regulates Klotho expression. Moreover, we found CIP significantly enhanced the expression of STAT4. Knockdown STAT4 suppressed Klotho up-regulation after CIP and blocked the CIP-induced brain ischemic tolerance. Collectively, it can be concluded that STAT4-mediated the up-regulation of Klotho contributed to the brain ischemic tolerance induced by CIP via inhibiting pyroptosis.

The Regulation of Autophagy by p38 MAPK-PPARγ Signaling During the Brain Ischemic Tolerance Induced by Cerebral Ischemic Preconditioning.

Several studies indicated that autophagy activation participates in brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIP). However, the mechanism of autophagy activation during the process still remains unclear. The present study aimed to evaluate the role of p38 MAPK-peroxisome proliferator-activated receptor γ (PPARγ) signaling cascade in autophagy during the CIP-induced BIT. The results shown that, initially, autophagy activation was observed after CIP in the model of global cerebral ischemia in rats, as was indicated by the upregulation of Beclin 1 expression, an increase in LC3-II/LC3-I ratio, the enhanced LC3 immunofluorescence, and a rise in the number of autophagosomes in the neurons of the hippocampal CA1 area. Besides, the inhibitor of autophagy 3-methyladenine obliterated the neuroprotection induced by CIP. Furthermore, the upregulation of p-p38 MAPK and PPARγ expressions was earlier than autophagy activation after CIP. In addition, pretreatment with SB203580 (the inhibitor of p38 MAPK) reversed CIP-induced PPARγ upregulation, autophagy activation, and neuroprotection. Pretreatment with GW9662 (the inhibitor of PPARγ) reversed autophagy activation and neuroprotection, while it had no effect on p-p38 MAPK upregulation induced by CIP. These data suggested that the p38 MAPK-PPARγ signaling pathway participates in autophagy activation during the induction of BIT by CIP.

Klotho Upregulation via PPARγ Contributes to the Induction of Brain Ischemic Tolerance by Cerebral Ischemic Preconditioning in Rats.

Cerebral ischemic preconditioning (CIP)-induced brain ischemic tolerance protects neurons from subsequent lethal ischemic insult. However, the specific mechanisms underlying CIP remain unclear. In the present study, we explored the hypothesis that peroxisome proliferator-activated receptor gamma (PPARγ) participates in the upregulation of Klotho during the induction of brain ischemic tolerance by CIP. First we investigated the expression of Klotho during the brain ischemic tolerance induced by CIP. Lethal ischemia significantly decreased Klotho expression from 6h to 7days, while CIP significantly increased Klotho expression from 12h to 7days in the hippocampal CA1 region. Inhibition of Klotho expression by its shRNA blocked the neuroprotection induced by CIP. These results indicate that Klotho participates in brain ischemic tolerance by CIP. Furthermore, we tested the role of PPARγ in regulating Klotho expression after CIP. CIP caused PPARγ protein translocation to the nucleus in neurons in the CA1 region of the hippocampus. Pretreatment with GW9962, a PPARγ inhibitor, significantly attenuated the upregulation of Klotho protein and blocked the brain ischemic tolerance induced by CIP. Taken together, it can be concluded that Klotho upregulation via PPARγ contributes to the induction of brain ischemic tolerance by CIP.

0576 Impact of Obstructive Sleep Apnea on In-Hospital Mortality Due to Acute Myocardial Infarction - A Large Retrospective National Study of Veterans

Abstract Introduction Ischemic preconditioning (IPC) is a phenomenon that refers to multiple, brief periods of myocardial ischemia followed by reperfusion. This counterintuitive preconditioning is thought to provide cardioprotection from subsequent prolonged ischemic events. Obstructive sleep apnea (OSA) may lead to IPC by frequent, recurrent short episodes of hypoxia and sympathetic stimulation. We hypothesized in patients who were admitted with acute myocardial infarction (MI), the presence of OSA was associated with improved survival during the hospitalization, likely due to IPC. Methods This is a retrospective cohort study utilizing national Veterans Health Administration (VHA) records. The cohort includes patients from 1999-2020 with a hospitalization for MI as the principal diagnosis. We confirm the OSA diagnosis using ICD9/10 codes. The primary outcome was in-hospital mortality during the index admission for acute MI. We reported the odds ratio (OR) of in-hospital mortality between with-OSA and without-OSA using logistic regression. We adjusted the OR (aOR) by age, gender, race, ethnicity, BMI, and Charlson Comorbidity index. Results Out of 4,237,444 veterans with any sleep diagnosis, 76,359 patients were hospitalized with a diagnosis of MI. We observed 30,116 with OSA (age, 64±10; BMI, 33±7) and 43,480 without OSA (age, 68±12; BMI, 29±6). The aOR of in-patient mortality was lower in with-OSA (n = 333[1.1%]; aOR, 1.86; 95% CI, 1.63 to 2.12) compared to without-OSA (n = 1,102[2.5%]; aOR, 2.02; 95% CI, 1.78 to 2.30). After stratifying the patients based on previous history of MI, the results remained the same. Conclusion We found that in patients admitted with acute MI, the OSA cohort was associated with lower mortality when compared to non-OSA cohort. We speculate that these differences may be attributed to IPC likely due to repetitive, chronic episodes of hypoxia in obstructive sleep apnea. Further research is warranted to elicit the clinical implications of ischemic preconditioning and OSA. Support (If Any) Culebras, A. Exploring the trail between cerebral ischemic aggravation and ischemic preconditioning. Obstructive sleep apnea leads the way. Sleep Medicine, Volume 67 ,2020, p 276-277Salman LA, Shulman R, Cohen JB. Obstructive sleep apnea, hypertension, and cardiovascular risk: epidemiology, pathophysiology, and management. Curr Cardiol Rep 2020;22(2):6

LncRNA BIRF Promotes Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning Through Upregulating GLT-1 via Sponging miR-330-5p

Long noncoding RNAs (lncRNAs) play an important regulatory role in various diseases. However, the role of lncRNAs in brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIPC) is still unknown. The lncRNA profile of rat cortical astrocytes pretreated with ischemic preconditioning was analyzed by high-throughput sequencing. The results of Cell-Counting Kit-8 (CCK-8) assay showed that a novel lncRNA, NONRATT009133.2, which we referred to as brain ischemia–related factor (BIRF), was highly correlated with BIT. Through bioinformatics analysis, we predicted that BIRF, miR-330-5p, and GLT-1 (also named Slc1a2) might constitute a ceRNA regulatory network in the induction of BIT. We found that BIRF was upregulated by CIPC, which promoted GLT-1 expression and BIT induction. BIRF could directly bind to miR-330-5p. Furthermore, miR-330-5p directly targeted GLT-1, and miR-330-5p inhibited both GLT-1 expression and BIT induction in vitro and in vivo. Moreover, BIRF acts as a molecular sponge to competitively bind to miR-330-5p with GLT-1 mRNA, while the miR-330-5p inhibitor reversed all the effects of BIRF siRNA on GLT-1 expression and neuronal vitality. Taken together, our results demonstrate the important roles of the BIRF/miR-330-5p/GLT-1 axis in the induction of BIT by CIPC. BIRF may be a potentially effective therapeutic strategy against stroke injury.

The p38 MAPK/NF-κB pathway mediates GLT-1 up-regulation during cerebral ischemic preconditioning-induced brain ischemic tolerance in rats

Our previous finding suggests that p38 MAPK contributes to the GLT-1 upregulation during induction of brain ischemic tolerance by cerebral ischemic preconditioning (CIP), however, the underlying mechanism is still unclear. Here, we investigated the molecular mechanisms underlying the CIP-induced GLT-1 upregulation by using Western blotting, Co-immunoprecipitation (Co-IP), electrophoretic mobility shift assay (EMSA) and thionin staining in rat hippocampus CA1 subset. We found that application of BAY11-7082 (an inhibitor of NF-κB), or dihydrokainate (an inhibitor of GLT-1), or SB203580 (an inhibitor of p38 MAPK) could attenuate the CIP-induced neuronal protection in hippocampus CA1 region of rats. Moreover, CIP caused rapid activation of NF-κB, as evidenced by nuclear translocation of NF-κB p50 protein, which led to active p50/p65 dimer formation and increased DNA binding activity. GLT-1 was also increased after CIP. Pretreatment with BAY11-7082 blocked the CIP-induced GLT-1 upregulation. The above results suggest that NF-κB participates in GLT-1 up-regulation during the induction of brain ischemic tolerance by CIP. We also found that pretreatment with SB203580 caused significant reduction of NF-κB p50 protein in nucleus, NF-κB p50/p65 dimer nuclear translocation and DNA binding activity of NF-κB. Together, we conclude that p38 MAPK/NF-κB pathway participates in the mediation of GLT-1 up-regulation during the induction of brain ischemic tolerance induced by CIP.

Exosomes containing miR-451a is involved in the protective effect of cerebral ischemic preconditioning against cerebral ischemia and reperfusion injury.

AimTo study the role of exosomes in the protective effect of cerebral ischemic preconditioning (cerebral‐IPC) against cerebral I/R injury.MethodMouse models of cerebral‐IPC and MCAO/R were established as described previously, and their behavioral, pathological, and proteomic changes were analyzed. Neuro‐2a subjected to OGD/R were treated with exosomes isolated from the plasma of sham‐operated and cerebral‐IPC mice. The differentially expressed miRNAs between exosomes derived from sham‐operated (S‐exosomes) and preconditioned (IPC‐exosomes) mice were identified through miRNA array, and their targets were identified through database search. The control and OGD/R cells were treated with the IPC‐exosomes, miRNA mimic or target protein inhibitor, and their viability, oxidative, stress and apoptosis rates were measured. The activated pathways were identified by analyzing the levels of relevant proteins.ResultsCerebral‐IPC mitigated the cerebral injury following ischemia and reperfusion, and increased the number of plasma exosomes. IPC‐exosomes increased the survival of Neuro‐2a cells after OGD/R. The miR‐451a targeting Rac1 was upregulated in the IPC‐exosomes relative to S‐exosomes. The miR‐451a mimic and the Rac1 inhibitor NSC23766 reversed OGD/R‐mediated activation of Rac1 and its downstream pathways.ConclusionCerebral‐IPC ameliorated cerebral I/R injury by inducing the release of exosomes containing miR‐451a.

Review Cerebral Ischemic Tolerance and Preconditioning: Methods, Mechanisms, Clinical Applications, and Challenges.

Stroke is one of the leading causes of morbidity and mortality worldwide, and it is increasing in prevalence. The limited therapeutic window and potential severe side effects prevent the widespread clinical application of the venous injection of thrombolytic tissue plasminogen activator and thrombectomy, which are regarded as the only approved treatments for acute ischemic stroke. Triggered by various types of mild stressors or stimuli, ischemic preconditioning (IPreC) induces adaptive endogenous tolerance to ischemia/reperfusion (I/R) injury by activating a multitude cascade of biomolecules, for example, proteins, enzymes, receptors, transcription factors, and others, which eventually lead to transcriptional regulation and epigenetic and genomic reprogramming. During the past 30 years, IPreC has been widely studied to confirm its neuroprotection against subsequent I/R injury, mainly including local ischemic preconditioning (LIPreC), remote ischemic preconditioning (RIPreC), and cross preconditioning. Although LIPreC has a strong neuroprotective effect, the clinical application of IPreC for subsequent cerebral ischemia is difficult. There are two main reasons for the above result: Cerebral ischemia is unpredictable, and LIPreC is also capable of inducing unexpected injury with only minor differences to durations or intensity. RIPreC and pharmacological preconditioning, an easy-to-use and non-invasive therapy, can be performed in a variety of clinical settings and appear to be more suitable for the clinical management of ischemic stroke. Hoping to advance our understanding of IPreC, this review mainly focuses on recent advances in IPreC in stroke management, its challenges, and the potential study directions.

Sumoylation of sodium/calcium exchanger in brain ischemia and ischemic preconditioning

The small ubiquitin-like modifier (SUMO) conjugation (or SUMOylation) is a post-translational protein modification mechanism activated by different stress conditions that has been recently investigated in experimental models of cerebral ischemia. The expression of SUMOylation enzymes and substrates is not restricted to the nucleus, since they are present also in the cytoplasm and on plasma membrane and are involved in several physiological and pathological conditions.In the last decades, convincing evidence have supported the idea that the increased levels of SUMOylated proteins may induce tolerance to ischemic stress. In particular, it has been established that protein SUMOylation may confer neuroprotection during ischemic preconditioning.Considering the increasing evidence that SUMO can modify stability and expression of ion channels and transporters and the relevance of controlling ionic homeostasis in ischemic conditions, the present review will resume the main aspects of SUMO pathways related to the key molecules involved in maintenance of ionic homeostasis during cerebral ischemia and ischemic preconditioning, with a particular focus on the on Na+/Ca2+ exchangers.

Ethanol extracts from Ilex pubescens promotes cerebral ischemic tolerance via modulation of TLR4-MyD88/TRIF signaling pathway in rats

Ethnopharmacological relevancePubescent Holly Root is the dry root of Ilex pubescens Hook. et Arn. It is clinically using in the treatment for stroke and coronary artery disease. It remains unclear whether the ethanol extracts of Ilex pubescens(IPEE) treatment can promote cerebral ischemic tolerance (CIT) and exert endogenous neuroprotective effects and thus to alleviate the nerve injury caused by the subsequent persistent cerebral ischemic attacks. Aim of the studyTo investigate the effects of IPEE on CIT and its underlying molecular mechanisms. Materials and methods: Adult male Wistar rats were used in the present study. The bilateral common carotid arteries were blocked for 10 min followed a subsequent reperfusion to create the cerebral ischemic preconditioning (CIP); After 3 days post CIP, rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R)-injury. Rats were continuously fed with IPEE for 5 days throughout the experiment period at the dose of 100 mg/kg and 200 mg/kg, respectively. Then, the brain infarct volume, histopathology, neurological deficits, and the gene/protein expression related with the TLR4-MyD88/TRIF signaling pathway were evaluated after 24 h of MCAO/R experiment. ResultsIPEE pretreatment significantly reduced the cerebral infarct volume, the neurological deficit scores, and the plasma level of neuron specific enolase (NSE) at the dose of 100 mg/kg. Meanwhile, IPEE pretreatment significantly decreased the levels of inflammatory cytokines including TNF-α, IL-6, MCP-1, MIP-1α and RANTES, while it increased the levels of anti-inflammatory cytokines, such as IL-10 and TGF-β, when compared with the group with CIP treatment alone. Moreover, the effect of IPEE treatment on CIT was in a dose-dependent manner, showing as a better effect in the group pretreated with IPEE with the dose of 100 mg/kg than that in group pretreated with IPEE with the dose of 200 mg/kg. In addition, IPEE pretreatment significantly inhibited the expressions of MyD88 mRNA and the protein expression of COX-2 and NF-κBp65, while it strengthened the expressions of TRIF mRNA and protein. The effects of IPEE pretreatment on the expression of these genes were better than that in the group treated with CIP alone. ConclusionsThe present study demonstrates that IPEE pretreatment can enhance cerebral ischemic tolerance with a underlying mechanism involved in the toll-like receptor 4 (TLR4) signaling pathway through inhibiting the production of proteins or cytokines in the downstream of MyD88 and activating TRIF dependent anti-inflammatory pathways.

Autophagy and Ischemic Stroke.

Ischemic stroke refers to brain tissue ischemia, hypoxic necrosis, and brain softening caused by the interruption of the blood supply to the brain without adequate collateral circulation, thus resulting in neurological symptoms. Autophagy is activated in various cell types in the brain, such as neurons, glial cells, and microvascular cells, upon ischemic stroke. Autophagy efflux injury plays an important role in this pathologic process. This chapter outlines the induction of basal autophagy, autophagy in neurons, and the crosstalk between autophagy, necrosis, and apoptosis that contributes to ischemic stroke. We will highlight the interactions between autophagy, oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction, and the role of autophagy in ischemic stroke. We will also review the recent advances in the understanding of the involvement of autophagy in the pathological process of cerebral ischemic preconditioning, periconditioning, and postconditioning.

Differential Expression of CXCL1, CXCL10, and CXCL12 in Response to Cerebral Ischemic Postconditioning in Rat Brain

Objectives: Cerebral ischemic preconditioning lessens stroke-induced injuries, but it is clinically feasible only when the occurrence of stroke is predictable. Reperfusion plays a critical role against cerebral injury after stroke; we tested the hypothesis that interrupting ischemia during early reperfusion, i.e. Postconditioning (POCO) affects CXC chemokine expression and further reduce inflammation in rat model of ischemia/reperfusion. Materials & Methods: Adult male Wistar rats (250-300 g) were used in this experiment. Using 4-vessel occlusion method, global cerebral ischemia was induced and POCO was performed by applying 3 cycles of 15-s/15-s reperfusion/reocclusion after a 45-s reperfusion (POCO-45-15/15). Western blotting analysis was used to investigate CXCL1, CXCL10 and CXCL12 expression 24 h, 48 h and one week after ischemic postconditioning (iPOCO). Results: Based on the results, iPOCO attenuates the expression of inflammatory chemokines CXCL1 and CXCL10 in hippocampus area of postconditioned rats, while the CXCL12 was not affected by iPOCO. Conclusion: Current findings may support chemokines role in iPOCO via reduction of inflammation. Also there could be a link between postconditioning, stress and inflammation through chemokines.

Preactivation of Notch1 in remote ischemic preconditioning reduces cerebral ischemia-reperfusion injury through crosstalk with the NF-\u03baB pathway

BackgroundRemote ischemic preconditioning (RIPC) initiates endogenous protective pathways in the brain from a distance and represents a new, promising paradigm in neuroprotection against cerebral ischemia-reperfusion (I/R) injury. However, the underlying mechanism of RIPC-mediated cerebral ischemia tolerance is complicated and not well understood. We reported previously that preactivation of Notch1 mediated the neuroprotective effects of cerebral ischemic preconditioning in rats subjected to cerebral I/R injury. The present study seeks to further explore the role of crosstalk between the Notch1 and NF-κB signaling pathways in the process of RIPC-induced neuroprotection.MethodsMiddle cerebral artery occlusion and reperfusion (MCAO/R) in adult male rats and oxygen-glucose deprivation and reoxygenation (OGD/R) in primary hippocampal neurons were used as models of I/R injury in vivo and in vitro, respectively. RIPC was induced by a 3-day procedure with 4 cycles of 5 min of left hind limb ischemia followed by 5 min of reperfusion each day before MCAO/R. Intracerebroventricular DAPT injection and sh-Notch1 lentivirus interference were used to inhibit the Notch1 signaling pathway in vivo and in vitro, respectively. After 24 h of reperfusion, neurological deficit scores, infarct volume, neuronal apoptosis, and cell viability were assessed. The protein expression levels of NICD, Hes1, Phospho-IKKα/β (p-IKK α/β), Phospho-NF-κB p65 (p-NF-κB p65), Bcl-2, and Bax were assessed by Western blotting.ResultsRIPC significantly improved neurological scores and reduced infarct volume and neuronal apoptosis in rats subjected to I/R injury. OGD preconditioning significantly reduced neuronal apoptosis and improved cell viability after I/R injury on days 3 and 7 after OGD/R. However, the neuroprotective effect was reversed by DAPT in vivo and attenuated by Notch1-RNAi in vitro. RIPC significantly upregulated the expression of proteins related to the Notch1 and NF-κB pathways. NF-κB signaling pathway activity was suppressed by a Notch1 signaling pathway inhibitor and Notch1-RNAi.ConclusionsThe neuroprotective effect of RIPC against cerebral I/R injury was associated with preactivation of the Notch1 and NF-κB pathways in neurons. The NF-κB pathway is a downstream target of the Notch1 pathway in RIPC and helps protect focal cerebral I/R injury.

Peroxisome proliferator‐activated receptor gamma participates in the acquisition of brain ischemic tolerance induced by ischemic preconditioning via glial glutamate transporter 1 in vivo and in vitro

Glial glutamate transporter 1 (GLT-1) plays a vital role in the induction of brain ischemic tolerance (BIT) by ischemic preconditioning (IPC). However, the mechanism still needs to be further explained. The aim of this study was to investigate whether peroxisome proliferator-activated receptor gamma (PPARγ) participates in regulating GLT-1 during the acquisition of BIT induced by IPC. Initially, cerebral IPC induced BIT and enhanced PPARγ and GLT-1 expression in the CA1 hippocampus in rats. The ratio of nuclear/cytoplasmic PPARγ was also increased. At the same time, the up-regulation of PPARγ expression in astrocytes in the CA1 hippocampus was revealed by double immunofluorescence for PPARγ and glial fibrillary acidic protein. Then, the mechanism by which PPARγ regulates GLT-1 was studied in rat cortical astrocyte-neuron cocultures. We found that IPC [45min of oxygen glucose deprivation (OGD)] protected neuronal survival after lethal OGD (4h of OGD), which usually leads to neuronal death. The activation of PPARγ occurred earlier than the up-regulation of GLT-1 in astrocytes after IPC, as determined by western blot and immunofluorescence. Moreover, the preadministration of the PPARγ antagonist T0070907 or PPARγ siRNA significantly attenuated GLT-1 up-regulation and the neuroprotective effects induced by IPC in vitro. Finally, the effect of the PPARγ antagonist on GLT-1 expression and BIT was verified in vivo. We observed that the preadministration of T0070907 by intracerebroventricular injection dose-dependently attenuated the up-regulation of GLT-1 and BIT induced by cerebral IPC in rats. In conclusion, PPARγ participates in regulating GLT-1 during the acquisition of BIT induced by IPC. Cover Image for this issue: doi: 10.1111/jnc.14532. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Profiling the Gene Expression and DNA Methylation in the Mouse Brain after Ischemic Preconditioning

Ischemic preconditioning (IPC) is a phenomenon in which a short-term sublethal ischemic exposure induces tolerance to a subsequent lethal ischemic insult; however, the detailed mechanism underlying IPC-induced neuroprotection remains obscure. Here, we applied middle cerebral artery occlusion, a preconditioning ischemic insult mouse model, to investigate the molecular mechanism underlying cerebral IPC. RNA sequencing and whole-genome bisulfite sequencing were performed to explore the gene expression profile and DNA methylation changes after cerebral IPC treatment. In this study, we identified 636 differentially expressed genes enriched for several pathways that were partially overlapping or interconnected in terms of similar gene function. The involvement of several genes in IPC-induced neuroprotection was first reported. Genes induced by IPC, including Arid5a, Nptx2 and Stc2, demonstrated a neuroprotective effect against oxygen–glucose deprivation induced neurotoxicity in vitro. Thus, our findings provide new insights into IPC signaling pathways and offer a novel therapeutic strategy towards stroke.

Metabolomic Profiling Reveals That Reprogramming of Cerebral Glucose Metabolism Is Involved in Ischemic Preconditioning-Induced Neuroprotection in a Rodent Model of Ischemic Stroke

Ischemic tolerance renders the brain resistant to ischemia-reperfusion (I/R) injury as a result of the activation of endogenous adaptive responses triggered by various types of preconditioning. The complex underlying metabolic mechanisms responsible for the neuroprotection of cerebral ischemic preconditioning (IPC) remain elusive. Herein, gas chromatography-mass spectrometry (GC-MS) technique was applied to delineate the dynamic changes of brain metabolome in a rodent model of ischemic stroke (transient occlusion of the middle cerebral artery, tMCAO), alone or after pretreatment with nonlethal ischemic tolerance induction (transient occlusion of the bilateral common carotid arteries, tBCCAO). Metabolomic analysis showed that accumulation of glucose (concentration increased more than 4 fold) and glycolytic intermediates is the prominent feature of brain I/R-induced metabolic disturbance. IPC attenuated brain I/R damage by subduing postischemic hyperglycolysis, increasing the pentose phosphate pathway (PPP) flux and promoting the utilization of β-hydroxybutyrate. The expression analysis of pivotal genes and proteins involved in relevant metabolic pathways revealed that the downregulation of AMP-activated protein kinase (AMPK)-mediated glucose transporter-1 (GLUT-1) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) and reduced mRNA levels of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits were associated with IPC-induced metabolic flexibility, which allows the brain to be more capable of withstanding severe I/R insults. The present study provided mechanistic insights into the metabolic signature of IPC and indicated that adaptively modulating brain glucose metabolism could be an effective approach for the therapeutic intervention of ischemic stroke.

Neuroprotective Effects of Cerebral Ischemic Preconditioning in a Rat Middle Cerebral Artery Occlusion Model: The Role of the Notch Signaling Pathway.

Cerebral ischemia-reperfusion (I/R) injury is a major problem worldwide. The Notch signaling pathway plays an important role in neural progenitor cell differentiation and in the inflammatory response after central nervous system injury. This study evaluated whether the neuroprotective effect of cerebral ischemic preconditioning (cIPC) is mediated by the preactivation of the Notch signaling pathway. A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model and glucose deprivation/reoxygenation (OGD/R) cell model were constructed to detect the neuroprotective effects of cIPC. In in vivo experiments, cIPC reduces the neurological functional deficit, cerebral infarction, and cellular apoptosis in the hippocampus induced by middle cerebral artery occlusion/reperfusion (MCAO/R), thus indicating that cIPC can improve neurologic function. Moreover, cIPC can reveal the expression peak of Jagged1, Notch1, NICD, and Hes1 protein, thereby indicating that cIPC can preactivate Notch signaling. However, cIPC-induced improvements in neurologic function are compromised by the γ-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-1-alanyl)-S-phenylglycine t-butyl ester (DAPT). In in vitro experiments, OGD preconditioning (OGDPC) can clearly upregulate Notch1 expression in the OGD/R-treated neuron and neural stem cell. Notch1 pre-overexpression can decrease neuron death and apoptosis under OGD/R treatment. Notch1 pre-overexpression can decrease the percentage of G1 stage cells and increase the percentage of S stage cells in OGD/R-treated neural stem cell. Furthermore, Notch1 pre-knockdown has the opposite effect on cell survival, apoptosis, and cycle in both OGD/R-treated neuron and neural stem cell. In conclusion, our results demonstrate that the neuroprotective effects of cIPC in a rat MCAO/R model are mediated by the preactivation of the Notch signaling pathway.

Ischemic preconditioning protects brain from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis through PERK pathway.

The purpose of this study was to explore the effects of cerebral ischemic preconditioning which can decrease brain ischemia/reperfusion (I/R) injury by inhibiting endoplasmic reticulum (ER) stress-induced apoptosis. The focal cerebral ischemia rat was selected as the experimental model. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells in ischemic penumbra were assessed after cerebral reperfusion. We assessed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells and measured the expressions of phosphorylation PERK (p-PERK), glucose-regulated protein 78 (GRP78), activating transcription factor-4 (ATF4) and caspase-12 in ischemic penumbra after cerebral reperfusion. We showed that the infarct sizes can be reduced due to the preconditioning under the influence of brain ischemia after reperfusion. The effect of preconditioning on the expression of ER stress proteins suggested the expressions of the 4 proteins p-PERK, ATF4, caspase-12 and GRP78 in the penumbra cortex by immunohistochemistry and Western blot increased after cerebral ischemia. Significant reduction of the number of TUNEL-positive cells was in the penumbra cortex of the preconditioning group. We found that cerebral ischemic preconditioning can protect the brain from I/R injury by inhibiting ER stress-induced apoptosis; the pathway of PERK is involved.

Stimulated CB1 Cannabinoid Receptor Inducing Ischemic Tolerance and Protecting Neuron from Cerebral Ischemia.

It is reported that endogenous cannabinoids can cause vasodilation and bradycardia. They have anti-inflammatory effect and protect endothelial cells from injury, therefore they have potential application prospect in the prevention of cardio-cerebrovascular diseases. However, the mechanisms of the neuroprotection mediated by cannabinoid 1 receptors (CB1Rs) have not been uncovered in detail. Nearly one hundred of new publications relevant to the theme are almost selected from Pubmed. The advanced details associated with the involvement of CB1R in cerebral ischemia as well as cerebral ischemic tolerance are reviewed. Anandamide system is mainly made up of cannabinoid receptors, their endogenous ligands and some related enzymes. The activation of the system mediates various molecular events so that plays a crucial role in the neuroprotection of cerebral ischemia. Increasing evidences suggest that CB1R is one of key molecules that mediate cerebral ischemia and cerebral ischemia tolerance. It is likely to provide an appropriate antioxidant balance by increasing endogenous free radical scavengers and helpful to exert the neuroprotective effects. Moreover, MAPKs, including ERK1/2, c-Jun Nterminal kinase (JNK) and p38MAPK can be recruited and stimulated through a complex signaling networks mediated by CB1R. Considerable evidences have indicated that CB1R was a crucial regulator for ERK1/2 signaling pathway. It is known that PI3K/Akt is a classical signaling pathway and its activation exerts neuroprotective effect via significant promoting cell survival. Glycogen synthase kinase-3β (GSK-3β) is an important downstream target of p-Akt. The PI3K/Akt/GSK-3β signaling pathway mediated by CB1Rs takes an important part in cerebral ischemic injury. PKC and CB1R are found to be abundantly co-expressed in presynaptic nerve endings of brain. There are considerable reports that different PKC isozymes played vital roles respectively in cerebral ischemic injury and preconditioning. The CB1R -mediated activation of PKCε can effectively stimulate ischemic tolerance. CB1R played an important part via several signaling pathways in the protection from ischemic stroke and in ischemic tolerance. The involved molecular signaling pathways include ERK1/2, PI3K/Akt/GSK-3β and the translocation and activation of PKCε. With the intimate association between CB1R and neuron injuries, to target the receptor will exert neuroprotective effects on cerebral ischemia, which provides wide foreground for a novel therapy target.