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1.  GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke 
The mood stabilizer lithium inhibits glycogen synthase kinase-3 (GSK-3) directly or indirectly by enhancing serine phosphorylation of both α and β isoforms. Lithium robustly protected primary brain neurons from glutamate-induced excitotoxicity; these actions were mimicked by other GSK-3 inhibitors or silencing/inhibiting GSK-3α and/or β isoforms. Lithium rapidly activated Akt to enhance GSK-3 serine phosphorylation and to block glutamate-induced Akt inactivation. Lithium also up-regulated Bcl-2 and suppressed glutamate-induced p53 and Bax. Induction of brain-derived neurotrophic factor (BDNF) was required for lithium’s neuroprotection to occur. BDNF promoter IV was activated by GSK-3 inhibition using lithium or other drugs, or through gene silencing/inactivation of either isoform. Further, lithium’s neuroprotective effects were associated with inhibition of NMDA receptor-mediated calcium influx and down-stream signaling. In rodent ischemic models, post-insult treatment with lithium decreased infarct volume, ameliorated neurological deficits, and improved functional recovery. Up-regulation of heat-shock protein 70 and Bcl-2 as well as down-regulation of p53 likely contributed to lithium’s protective effects. Delayed treatment with lithium improved functional MRI responses, which was accompanied by enhanced angiogenesis. Two GSK-3-regulated pro-angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor were induced by lithium. Finally, lithium promoted migration of mesenchymal stem cells (MSCs) by up-regulation of MMP-9 through GSK-3β inhibition. Notably, transplantation of lithium-primed MSCs into ischemic rats enhanced MSC migration to the injured brain regions and improved the neurological performance. Several other GSK-3 inhibitors have also been reported to be beneficial in rodent ischemic models. Together, GSK-3 inhibition is a rational strategy to combat ischemic stroke and other excitotoxicity-related brain disorders.
doi:10.3389/fnmol.2011.00015
PMCID: PMC3152742  PMID: 21886605
lithium; glycogen synthase kinase-3; excitotoxicity; cerebral ischemia; mesenchymal stem cells
2.  Lithium Ameliorates Neurodegeneration, Suppresses Neuroinflammation, and Improves Behavioral Performance in a Mouse Model of Traumatic Brain Injury 
Journal of Neurotrauma  2012;29(2):362-374.
Abstract
Although traumatic brain injury (TBI) is recognized as one of the leading causes of death from trauma to the central nervous system (CNS), no known treatment effectively mitigates its effects. Lithium, a primary drug for the treatment of bipolar disorder, has been known to have neuroprotective effects in various neurodegenerative conditions such as stroke. Until this study, however, it has not been investigated as a post-insult treatment for TBI. To evaluate whether lithium could have beneficial effects following TBI, lithium at a dose of 1.5 mEq/kg was administered after injury. Assessed at 3 days and 3 weeks post-injury using hematoxylin and eosin staining, lithium treatment was found to reduce lesion volume. Lithium at doses of 2.0 and 3.0 mEq/kg also significantly reduced lesion volume at 3 days after injury, and the therapeutic window was at least 3 h post-injury. TBI-induced neuronal death, microglial activation, and cyclooxygenase-2 induction were all attenuated by lithium at 3 days after injury. In addition, lithium treatment reduced TBI-induced matrix metalloproteinase-9 expression and preserved the integrity of the blood–brain barrier. As for behavioral outcomes, lithium treatment reduced anxiety-like behavior in an open-field test, and improved short- and long-term motor coordination in rotarod and beam-walk tests. Lithium robustly increased serine phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that the underlying mechanisms responsible for lithium's protective effects are triggered by increasing phosphorylation of this kinase and thereby inhibiting its activity. Our results support the notion that lithium has heretofore unrecognized capacity to mitigate the neurodegenerative effects and improve functional outcomes in TBI.
doi:10.1089/neu.2011.1942
PMCID: PMC3261788  PMID: 21895523
GSK-3β; lithium; neuroinflammation; neuroprotection; traumatic brain injury
3.  Involvement of the Glycogen Synthase Kinase-3 Signaling Pathway in TBI Pathology and Neurocognitive Outcome 
PLoS ONE  2011;6(9):e24648.
Background
Traumatic brain injury (TBI) sets in motion cascades of biochemical changes that result in delayed cell death and altered neuronal architecture. Studies have demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) effectively reduces apoptosis following a number of stimuli. The Wnt family of proteins, and growth factors are two major factors that regulate GSK-3 activity. In the absence of stimuli, GSK-3 is constitutively active and is complexed with Axin, adenomatous polyposis coli (APC), and casein kinase Iα (CK1α) and phosphorylates ß-Catenin leading to its degradation. Binding of Wnt to Frizzled receptors causes the translocation of GSK-3 to the plasma membrane, where it phosphorylates and inactivates the Frizzled co-receptor lipoprotein-related protein 6 (LRP6). Furthermore, the translocation of GSK-3 reduces ß-Catenin phosphorylation and degradation, leading to ß-Catenin accumulation and gene expression. Growth factors activate Akt, which in turn inhibits GSK-3 activity by direct phosphorylation, leading to a reduction in apoptosis.
Methodology/Principal Findings
Using a rodent model, we found that TBI caused a rapid, but transient, increase in LRP6 phosphorylation that is followed by a modest decrease in ß-Catenin phosphorylation. Phospho-GSK-3β immunoreactivity was found to increase three days post injury, a time point at which increased Akt activity following TBI has been observed. Lithium influences several neurochemical cascades, including inhibiting GSK-3. When the efficacy of daily lithium was assessed, reduced hippocampal neuronal cell loss and learning and memory improvements were observed. These influences were partially mimicked by administration of the GSK-3-selective inhibitor SB-216763, as this drug resulted in improved motor function, but only a modest improvement in memory retention and no overt neuroprotection.
Conclusion/Significance
Taken together, our findings suggest that selective inhibition of GSK-3 may offer partial cognitive improvement. As a broad spectrum inhibitor of GSK-3, lithium offers neuroprotection and robust cognitive improvement, supporting its clinical testing as a treatment for TBI.
doi:10.1371/journal.pone.0024648
PMCID: PMC3174188  PMID: 21935433
4.  Inhibition of Glycogen Synthase Kinase-3β Improves Tolerance to Ischemia in Hypertrophied Hearts 
The Annals of thoracic surgery  2007;84(1):126-133.
Background
Hypertrophied myocardium is more susceptible to ischemia/reperfusion injury, in part owing to impaired insulin-mediated glucose uptake. Glycogen synthase kinase-3β (GSK-3β) is a key regulatory enzyme in glucose metabolism that, when activated, phosphorylates/inactivates target enzymes of the insulin signaling pathway. Glycogen synthase kinase-3β is regulated upstream by Akt-1. We sought to determine whether GSK-3β is activated in ischemic hypertrophied myocardium owing to impaired Akt-1 function, and whether inhibition with lithium (Li) or indirubin-3′-monoxime,5-iodo- (IMI), a specific inhibitor, improves post-ischemic myocardial recovery by improving glucose metabolism.
Methods
Pressure-overload hypertrophy was achieved by aortic banding in neonatal rabbits. At 6 weeks, isolated hypertrophied hearts underwent 30 minutes of normothermic ischemia and reperfusion with or without GSK-3β inhibitor (0.1 mM Li; 1 µM IMI) as cardioplegic additives. Cardiac function was measured before and after ischemia. Expression, activity of Akt-1 and GSK-3β, and lactate were determined at end-ischemia.
Results
Contractile function after ischemia was better preserved in hypertrophied hearts treated with GSK-3β inhibitors. Activity of Akt-1 was significantly impaired in hypertrophied myocardium at end-ischemia. Glycogen synthase kinase-3β enzymatic activity at end-ischemia was increased in hypertrophied hearts and was blocked by Li or IMI concomitant with significantly increased lactate production, indicating increased glycolysis.
Conclusions
Regulatory inhibition of GSK-3β by Akt-1 in hypertrophied hearts is impaired, leading to activation during ischemia. Inhibition of GSK-3β by Li or IMI improves tolerance to ischemia/reperfusion injury in hypertrophied myocardium. The likely protective mechanism is an increase in insulin-mediated glucose uptake, resulting in greater substrate availability for glycolysis during ischemia and early reperfusion.
doi:10.1016/j.athoracsur.2007.02.015
PMCID: PMC3444259  PMID: 17588398
5.  Post-ischemic estradiol treatment reduced glial response and triggers distinct cortical and hippocampal signaling in a rat model of cerebral ischemia 
Background
Estradiol has been shown to exert neuroprotective effects in several neurodegenerative conditions, including cerebral ischemia. The presence of this hormone prior to ischemia attenuates the damage associated with such events in a rodent model (middle cerebral artery occlusion (MCAO)), although its therapeutic value when administered post-ischemia has not been assessed. Hence, we evaluated the effects of estradiol treatment after permanent MCAO (pMCAO) was induced in rats, studying the PI3K/AKT/GSK3/β-catenin survival pathway and the activation of SAPK-JNK in two brain areas differently affected by pMCAO: the cortex and hippocampus. In addition, we analyzed the effect of estradiol on the glial response to injury.
Methods
Male rats were subjected to pMCAO and estradiol (0.04 mg/kg) was administered 6, 24, and 48 h after surgery. The animals were sacrificed 6 h after the last treatment, and brain damage was evaluated by immunohistochemical quantification of ‘reactive gliosis’ using antibodies against GFAP and Iba1. In addition, Akt, phospho-AktSer473, phospho-AktThr308, GSK3, phospho-GSK3Ser21/9, β-catenin, SAPK-JNK, and pSAPK-JNKThr183/Tyr185 levels were determined in western blots of the ipsilateral cerebral cortex and hippocampus, and regional differences in neuronal phospho-Akt expression were determined by immunohistochemistry.
Results
The increases in the percentage of GFAP- (5.25-fold) and Iba1- (1.8-fold) labeled cells in the cortex and hippocampus indicate that pMCAO induced ‘reactive gliosis’. This effect was prevented by post-ischemic estradiol treatment; diminished the number of these cells to those comparable with control animals. pMCAO down-regulated the PI3K/AkT/GSK3/β-catenin survival pathway to different extents in the cortex and hippocampus, the activity of which was restored by estradiol treatment more efficiently in the cerebral cortex (the most affected region) than in the hippocampus. No changes in the phosphorylation of SAPK-JNK were observed 54 h after inducing pMCAO, whereas pMCAO did significantly decrease the phospho-AktSer473 in neurons, an effect that was reversed by estradiol.
Conclusion
The present study demonstrates that post-pMCAO estradiol treatment attenuates ischemic injury in both neurons and glia, events in which the PI3K/AKT/GSK3/β-catenin pathway is at least partly involved. These findings indicate that estradiol is a potentially useful treatment to enhance recovery after human ischemic stroke.
doi:10.1186/1742-2094-9-157
PMCID: PMC3414748  PMID: 22747981
MCAO; Focal ischemia; Rat; Estradiol; Brain; Estrogen; Neuroprotection; Stroke; Western blot; Immunohistochemistry; Akt
6.  Activation of Akt/protein kinase B mediates the protective effects of mechanical stretching against myocardial ischemia-reperfusion injury 
Journal of Veterinary Science  2012;13(3):235-244.
Akt/protein kinase B is a well-known cell survival factor and activated by many stimuli including mechanical stretching. Therefore, we evaluated the cardioprotective effect of a brief mechanical stretching of rat hearts and determined whether activation of Akt through phosphatidylinositol 3-kinase (PI3K) is involved in stretch-induced cardioprotection (SIC). Stretch preconditioning reduced infarct size and improved post-ischemic cardiac function compared to the control group. Phosphorylation of Akt and its downstream substrate, GSK-3β, was increased by mechanical stretching and completely blocked by wortmannin, a PI3K inhibitor. Treatment with lithium or SB216763 (GSK-3β inhibitors) before ischemia induction mimicked the protective effects of SIC on rat heart. Gadolinium (Gd3+), a blocker of stretch-activated ion channels (SACs), inhibited the stretch-induced phosphorylation of Akt and GSK-3β. Furthermore, SIC was abrogated by wortmannin and Gd3+. In vivo stretching induced by an aorto-caval shunt increased Akt phosphorylation and reduced myocardial infarction; these effects were diminished by wortmannin and Gd3+ pretreatment. Our results showed that mechanical stretching can provide cardioprotection against ischemia-reperfusion injury. Additionally, the activation of Akt, which might be regulated by SACs and the PI3K pathway, plays an important role in SIC.
doi:10.4142/jvs.2012.13.3.235
PMCID: PMC3467398  PMID: 23000580
Akt/protein kinase B; cardioprotection; ischemia-reperfusion injury; mechanical stretching
7.  Docosahexaenoic Acid Signaling Modulates Cell Survival in Experimental Ischemic Stroke Penumbra and Initiates Long-Term Repair in Young and Aged Rats 
PLoS ONE  2012;7(10):e46151.
Background
Docosahexaenoic acid, a major omega-3 essential fatty acid family member, improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia. We hypothesize that DHA elicits neuroprotection by inducing AKT/p70S6K phosphorylation, which in turn leads to cell survival and protects against ischemic stroke in young and aged rats.
Methods and Results
Rats underwent 2 h of middle cerebral artery occlusion (MCAo). DHA, neuroprotectin D1 (NPD1) or vehicle (saline) was administered 3 h after onset of stroke. Neurological function was evaluated on days 1, 2, 3, and 7. DHA treatment improved functional recovery and reduced cortical, subcortical and total infarct volumes 7 days after stroke. DHA also reduced microglia infiltration and increased the number of astrocytes and neurons when compared to vehicle on days 1 and 7. Increases in p473 AKT and p308 AKT phosphorylation/activation were observed in animals treated with DHA 4 h after MCAo. Activation of other members of the AKT signaling pathway were also observed in DHA treated animals including increases in pS6 at 4 h and pGSK at 24 h. DHA or NPD1 remarkably reduced total and cortical infarct in aged rats. Moreover, we show that in young and aged rats DHA treatment after MCAo potentiates NPD1 biosynthesis. The phosphorylation of p308 AKT or pGSK was not different between groups in aged rats. However, pS6 expression was increased with DHA or NPD1 treatment when compared to vehicle.
Conclusions
We suggest that DHA induces cell survival, modulates the neuroinflammatory response and triggers long term restoration of synaptic circuits. Both DHA and NPD1 elicited remarkable protection in aged animals. Accordingly, activation of DHA signaling might provide benefits in the management of ischemic stroke both acutely as well as long term to limit ensuing disabilities.
doi:10.1371/journal.pone.0046151
PMCID: PMC3484151  PMID: 23118851
8.  Phosphorylation of tau protein over time in rats subjected to transient brain ischemia 
Neural Regeneration Research  2013;8(34):3173-3182.
Transient brain ischemia has been shown to induce hyperphosphorylation of the microtubule-associated protein tau. To further determine the mechanisms underlying these processes, we investigated the interaction between tau, glycogen synthase kinase (GSK)-3β and protein phos-phatase 2A. The results confirmed that tau protein was dephosphorylated during brain ischemia; in addition, the activity of GSK-3β was increased and the activity of protein phosphatase 2A was decreased. After reperfusion, tau protein was hyperphosphorylated, the activity of GSK-3β was decreased and the activity of protein phosphatase 2A remained low. Importantly, the interaction of tau with GSK-3β and protein phosphatase 2A was altered during ischemia and reperfusion. Lithium chloride could affect tau phosphorylation by regulating the interaction of tau with GSK-3β and protein phosphatase 2A, and improve learning and memory ability of rats after transient brain ischemia. The present study demonstrated that it was the interaction of tau with GSK-3β and protein phosphatase 2A, rather than their individual activities, that dominates the phosphorylation of tau in transient brain ischemia. Hyperphosphorylated tau protein may play an important role in the evolution of brain injury in ischemic stroke. The neuroprotective effects of lithium chloride partly depend on the inhibition of tau phosphorylation during transient brain ischemia.
doi:10.3969/j.issn.1673-5374.2013.34.001
PMCID: PMC4146185  PMID: 25206638
neural regeneration; brain injury; brain ischemia; reperfusion; microtubule-associated protein tau; phosphorylation; glycogen synthase kinase 3β; protein phosphatase 2A; lithium chloride; grants-supported paper; neuroregeneration
9.  Altered Regulation of Akt Signaling with Murine Cerebral Malaria, Effects on Long-Term Neuro-Cognitive Function, Restoration with Lithium Treatment 
PLoS ONE  2012;7(10):e44117.
Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling likely underlies long-term neurological sequelae observed in ECM and may yield adjunctive therapeutic targets for the management of CM.
doi:10.1371/journal.pone.0044117
PMCID: PMC3474787  PMID: 23082110
10.  Lithium-mediated long-term neuroprotection in neonatal rat hypoxia-ischemia is associated with antiinflammatory effects and enhanced proliferation and survival of neural stem/progenitor cells 
The aim of this study was to evaluate the long-term effects of lithium treatment on neonatal hypoxic-ischemic brain injury, inflammation, and neural stem/progenitor cell (NSPC) proliferation and survival. Nine-day-old male rats were subjected to unilateral hypoxia-ischemia (HI) and 2 mmol/kg lithium chloride was injected intraperitoneally immediately after the insult. Additional lithium injections, 1 mmol/kg, were administered at 24-hour intervals for 7 days. Animals were killed 6, 24, 72 hours, or 7 weeks after HI. Lithium reduced total tissue loss by 69%, from 89.4±14.6 mm3 in controls (n=15) to 27.6±6.2 mm3 in lithium-treated animals (n=14) 7 weeks after HI (P<0.001). Microglia activation was inhibited by lithium treatment, as judged by Iba-1 and galectin-3 immunostaining, and reduced interleukin-1β and CCL2 levels. Lithium increased progenitor, rather than stem cell, proliferation in both nonischemic and ischemic brains, as judged by 5-bromo-2-deoxyuridine labeling 24 and 72 hours as well as by phospho-histone H3 and brain lipid-binding protein labeling 7 weeks after HI. Lithium treatment also promoted survival of newborn NSPCs, without altering the relative levels of neuronal and astroglial differentiation. In summary, lithium conferred impressive, morphological long-term protection against neonatal HI, at least partly by inhibiting inflammation and promoting NSPC proliferation and survival.
doi:10.1038/jcbfm.2011.75
PMCID: PMC3208156  PMID: 21587270
asphyxia; inflammation; microglia; neurogenesis; stem cell
11.  Pyruvate Protects Brain Against Ischemia-Reperfusion Injury by Activating Erythropoietin Signaling Pathway 
Background and Purpose
Pyruvate is known to be cytoprotective through antioxidant and anti-inflammatory mechanisms. We tested the hypothesis that pyruvate protects brain against ischemia-reperfusion injury by inducing endogenous erythropoietin (EPO) expression.
Methods
Pyruvate’s protective effect was evaluated in C6 glioma cells and HT22 neuronal cells subjected to transient oxygen glucose deprivation (OGD). Cell viability (calcein AM assay) and expression of HIF-1α, EPO, Akt and Erk (immunoblot) and EPO receptor (RT-PCR) were analyzed. Transient focal cerebral ischemia in rats was induced by 2h middle cerebral artery occlusion followed by 24h reperfusion. Pyruvate or saline was infused from 60 min occlusion until 30 min reperfusion. Lesion volume and DNA fragmentation were assessed by 2,3,5-triphenyltetrazolium staining and TUNEL assay, respectively. Immunoblots were conducted to determine cerebral EPO contents.
Results
Pyruvate increased cell viability, HIF-1α, EPO, and Akt phosphorylation. siRNA suppression of HIF-1α and EPO abolished pyruvate-induced cytoprotection. In the rat stroke model, pyruvate reduced lesion volume by 84% and DNA fragmentation by 77% vs. controls; increased EPO content paralleled these cerebroprotective actions of pyruvate.
Conclusions
Pyruvate activation of the HIF-1α-EPO signaling cascade in neurons and glia could protect brain from ischemia-reperfusion injury.
doi:10.1161/STROKEAHA.111.620088
PMCID: PMC3314723  PMID: 22282883
12.  Lithium Protection of Phencyclidine-induced Neurotoxicity in Developing Brain: the Role of PI-3K/Akt and MEK/ERK Signaling Pathways 
Phencyclidine (PCP) and other N-methyl-D-aspartate (NMDA) receptor antagonists have been shown to be neurotoxic to developing brains and to result in schizophrenia-like behaviors later in development. Prevention of both effects by antischizophrenic drugs suggests the validity of PCP neurodevelopmental toxicity as a heuristic model of schizophrenia. Lithium is used for the treatment of bipolar and schizoaffective disorders and has recently been shown to have neuroprotective properties. The present study used organotypic corticostriatal slices taken from postnatal day 2 rat pups to investigate the protective effect of lithium and the role of the phosphatidylinositol-3 kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/extracellular regulated kinase (MEK/ERK) pathways in PCP-induced cell death. Lithium pretreatment dose-dependently reduced PCP-induced caspase-3 activation and DNA fragmentation in layer II-IV of the cortex. PCP elicited time-dependent inhibition of the MEK/ERK and PI-3K/Akt pathways, as indicated by dephosphorylation of ERK1/2 and Akt. The pro-apoptotic factor glycogen synthase kinase-3β (GSK-3β) was also dephosphorylated at serine 9 and thus activated. Lithium prevented PCP-induced inhibition of the two pathways and activation of GSK-3β. Furthermore, blocking either PI-3K/Akt or MEK/ERK pathway abolished the protective effect of lithium, while inhibiting GSK-3β activity mimicked the protective effect of lithium. However, no crosstalk between the two pathways was found. Finally, specific GSK-3β inhibition did not prevent PCP-induced dephosphorylation of Akt and ERK. These data strongly suggest that the protective effect of lithium against PCP-induced neuroapoptosis is mediated through independent stimulation of the PI-3K/Akt and ERK pathways and suppression of GSK-3β activity.
doi:10.1124/jpet.107.133272
PMCID: PMC2561310  PMID: 18544676
13.  Ferulic acid regulates the AKT/GSK-3β/CRMP-2 signaling pathway in a middle cerebral artery occlusion animal model 
Laboratory Animal Research  2013;29(2):63-69.
Ferulic acid, a component of the plants Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort, exerts a neuroprotective effect by regulating various signaling pathways. This study showed that ferulic acid treatment prevents the injury-induced increase of collapsin response mediator protein 2 (CRMP-2) in focal cerebral ischemia. Glycogen synthase kinase-3β (GSK-3β) regulates CRMP-2 function through phosphorylation of CRMP-2. Moreover, the pro-apoptotic activity of GSK-3β is inactivated by phosphorylation by Akt. This study investigated whether ferulic acid modulates the expression of CRMP-2 and its upstream targets, Akt and GSK-3β, in focal cerebral ischemia. Male rats were treated immediately with ferulic acid (100 mg/kg, i.v.) or vehicle after middle cerebral artery occlusion (MCAO), and then cerebral cortices were collected 24 hr after MCAO. MCAO resulted in decreased levels of phospho-Akt and phospho-GSK-3β, while ferulic acid treatment prevented the decrease in the levels of these proteins. Moreover, phospho-CRMP-2 and CRMP-2 levels increased during MCAO, whereas ferulic acid attenuated these injury-induced increases. These results demonstrate that ferulic acid regulates the Akt/GSK-3β/CRMP-2 signaling pathway in focal cerebral ischemic injury, thereby protecting against brain injury.
doi:10.5625/lar.2013.29.2.63
PMCID: PMC3696626  PMID: 23825478
Ferulic acid; neuroprotection; Akt; GSK-3β; CRMP-2
14.  The Akt pathway is involved in rapid ischemic tolerance in focal ischemia in Rats 
Translational stroke research  2010;1(3):202-209.
Although the protective mechanisms of delayed ischemic preconditioning have received extensive studies, few have addressed the mechanisms associated with rapid ischemic postconditioning. We investigated whether ischemic tolerance induced by rapid preconditioning is regulated by the Akt survival signaling pathway. Stroke was generated by permanent occlusion of the left distal middle cerebral artery (MCA) plus 30 min or 1 h occlusion of the bilateral common carotid artery (CCA) in male rats. Rapid preconditioning performed 1h before stroke onset reduced infarct size by 69% in rats with 30 min CCA occlusion, but by only 19% with 1 h occlusion. After control ischemia with 30 min CCA occlusion, Western Blot showed that P-Akt was transiently increased while Akt kinase assay showed that Akt activity was decreased. Although preconditioning did not change P-Akt levels at 1h and 5h compared with control ischemia, it attenuated reduction in Akt activity at 5h in the penumbra. However, preconditioning did not change the levels of P-PDK1, P-PTEN, and P-GSK3β in the Akt pathway, all of which were decreased after stroke. At last, the PI3K kinase inhibitor, LY294002, completely reversed the protection from ischemic preconditioning. In conclusion, Akt contributes to the protection of rapid preconditionin against stroke.
doi:10.1007/s12975-010-0017-5
PMCID: PMC3144475  PMID: 21804899
rapid preconditioning; ischemic tolerance; cerebral ischemia; focal ischemia; neuroprotection; Akt
15.  Lithium-Mediated Protection Against Ethanol Neurotoxicity 
Lithium has long been used as a mood stabilizer in the treatment of manic-depressive (bipolar) disorder. Recent studies suggest that lithium has neuroprotective properties and may be useful in the treatment of acute brain injuries such as ischemia and chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. One of the most important neuroprotective properties of lithium is its anti-apoptotic action. Ethanol is a neuroteratogen and fetal alcohol spectrum disorders (FASD) are caused by maternal ethanol exposure during pregnancy. FASD is the leading cause of mental retardation. Ethanol exposure causes neuroapoptosis in the developing brain. Ethanol-induced loss of neurons in the central nervous system underlies many of the behavioral deficits observed in FASD. Excessive alcohol consumption is also associated with Wernicke–Korsakoff syndrome and neurodegeneration in the adult brain. Recent in vivo and in vitro studies indicate that lithium is able to ameliorate ethanol-induced neuroapoptosis. Lithium is an inhibitor of glycogen synthase kinase 3 (GSK3) which has recently been identified as a mediator of ethanol neurotoxicity. Lithium's neuroprotection may be mediated by its inhibition of GSK3. In addition, lithium also affects many other signaling proteins and pathways that regulate neuronal survival and differentiation. This review discusses the recent evidence of lithium-mediated protection against ethanol neurotoxicity and potential underlying mechanisms.
doi:10.3389/fnins.2010.00041
PMCID: PMC2907128  PMID: 20661453
alcohol; apoptosis; development; fetal alcohol syndrome; neurodegeneration; neuroprotection
16.  TLR2 ligand induces protection against cerebral ischemia/reperfusion injury: via activation of PI3K/Akt signaling 
This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n=8/group) were treated with Pam3CSK4 one hr before cerebral ischemia (60 min) followed by reperfusion (24 hrs). Pam3CSK4 was also given to the mice (n=8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p<0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4 treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, Bcl2 and decreased Bax levels and NF-κB binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3β/GSK-3β compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2 specific agonist during cerebral ischemia is effective in reducing injury.
doi:10.4049/jimmunol.1003428
PMCID: PMC3140609  PMID: 21709150
Apoptosis, Cerebral ischemia/reperfusion; Pam3CSK4; PI3K/Akt; TLR2
17.  Inhibition of GSK-3 Ameliorates Aβ Pathology in an Adult-Onset Drosophila Model of Alzheimer's Disease 
PLoS Genetics  2010;6(9):e1001087.
Aβ peptide accumulation is thought to be the primary event in the pathogenesis of Alzheimer's disease (AD), with downstream neurotoxic effects including the hyperphosphorylation of tau protein. Glycogen synthase kinase-3 (GSK-3) is increasingly implicated as playing a pivotal role in this amyloid cascade. We have developed an adult-onset Drosophila model of AD, using an inducible gene expression system to express Arctic mutant Aβ42 specifically in adult neurons, to avoid developmental effects. Aβ42 accumulated with age in these flies and they displayed increased mortality together with progressive neuronal dysfunction, but in the apparent absence of neuronal loss. This fly model can thus be used to examine the role of events during adulthood and early AD aetiology. Expression of Aβ42 in adult neurons increased GSK-3 activity, and inhibition of GSK-3 (either genetically or pharmacologically by lithium treatment) rescued Aβ42 toxicity. Aβ42 pathogenesis was also reduced by removal of endogenous fly tau; but, within the limits of detection of available methods, tau phosphorylation did not appear to be altered in flies expressing Aβ42. The GSK-3–mediated effects on Aβ42 toxicity appear to be at least in part mediated by tau-independent mechanisms, because the protective effect of lithium alone was greater than that of the removal of tau alone. Finally, Aβ42 levels were reduced upon GSK-3 inhibition, pointing to a direct role of GSK-3 in the regulation of Aβ42 peptide level, in the absence of APP processing. Our study points to the need both to identify the mechanisms by which GSK-3 modulates Aβ42 levels in the fly and to determine if similar mechanisms are present in mammals, and it supports the potential therapeutic use of GSK-3 inhibitors in AD.
Author Summary
Alzheimer's disease (AD) is the leading cause of dementia in the ageing population. Symptoms include memory loss and decline in understanding and reasoning. Alois Alzheimer, who reported the first case of AD, observed plaques and tangles in the brains of patients. The plaques are made up of amyloid protein, while the tangles are of tau protein. One of the main scientific ideas about AD is that it starts with build-up of amyloid, which then alters tau protein, causing the disease. Another protein, called GSK-3, also seems to play a part. Simple invertebrates such as flies are useful for understanding human diseases. We have created an AD model in the fruit fly Drosophila where amyloid protein is present in the nerve cells of the adult fly; this caused the flies to be impaired in their survival, nerve function, and behavior. We found that amyloid increased the activity of GSK-3, and so we experimentally turned down its activity and found that this improved the survival and behavior of the flies. Importantly, turning down the activity of GSK-3 in flies that did not have amyloid did not seem to harm them. GSK-3 could therefore be a good target for drugs against AD.
doi:10.1371/journal.pgen.1001087
PMCID: PMC2932684  PMID: 20824130
18.  Hypercholesterolemic Myocardium Is Vulnerable to Ischemia-Reperfusion Injury and Refractory to Sevoflurane-Induced Protection 
PLoS ONE  2013;8(10):e76652.
Recent studies have demonstrated that volatile anesthetic postconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia. Therefore, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. In the present study, normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane for 5 min or 3 cycles of 10-s ischemia/10-s reperfusion. The hemodynamic parameters, including left ventricular developed pressure, left ventricular end-diastolic pressure and heart rate, were continuously monitored. The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. We found that both sevoflurane and ischemic postconditioning significantly improved heart pump function, reduced infarct size and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β in the healthy rats. In the hypercholesterolemic rats, neither sevoflurane nor ischemic postconditioning improved left ventricular hemodynamics, reduced infarct size and increased the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. In conclusions, hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.
doi:10.1371/journal.pone.0076652
PMCID: PMC3790738  PMID: 24124583
19.  Ischemic postconditioning facilitates brain recovery after stroke by promoting Akt/mTOR activity in nude rats 
Journal of neurochemistry  2013;127(5):723-732.
While preconditioning is induced before stroke onset, ischemic postconditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in T-cell-deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post-stroke in wild type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p-Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p-mTOR, p-S6K and p-4EBP1 in the mTOR pathway, as well as GAP-43, both in the peri-infarct area and core, 24 hours after stroke. IPostC improved neurological function in nude rats 1–30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long-term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long-term protection by enhancing Akt and mTOR activity during the acute post-stroke phase.
doi:10.1111/jnc.12342
PMCID: PMC3875603  PMID: 23777415
Stroke; Ischemic postconditioning; T cells; Akt; mTOR
20.  Hypothermia Blocks β-catenin Degradation after Focal Ischemia in Rats 
Brain research  2008;1198:182-187.
Dephosphorylated and activated glycogen synthase kinase (GSK) 3β hyperphophorylates β-catenin, leading to its ubiquitin-proteosome-mediated degradation. β-catenin-knockdown increases while β-catenin overexpression prevents neuronal death in vitro; in addition, protein levels of β-catenin are reduced in the brain of Alzheimer’s patients. However, whether β-catenin degradation is involved in stroke-induced brain injury is unknown. Here we studied activities of GSK3 β and β-catenin, and the protective effect of moderate hypothermia (30 °C) on these activities after focal ischemia in rats. The results of Western blot showed that GSK3 β was dephosphorylated at 5 and 24 hours after stroke in the normothermic (37 °C) brain; hypothermia augmented GSK3β dephosphorylation. Because hypothermia reduces infarction, these results contradict with previous studies showing that GSK3β dephosphorylation worsens neuronal death. Nevertheless, hypothermia blocked degradation of total GSK3β protein. Corresponding to GSK3β activity in normothermic rats, β-catenin phosphorylation transiently increased at 5 hours in both the ischemic penumbra and core, and the total protein level of β-catenin degraded after normothermic stroke. Hypothermia did not inhibit β-catenin phosphorylation, but it blocked β-catenin degradation in the ischemic penumbra. In conclusion, moderate hypothermia can stabilize β-catenin, which may contribute to the protective effect of moderate hypothermia.
doi:10.1016/j.brainres.2008.01.007
PMCID: PMC2350209  PMID: 18241848
Focal ischemia; hypothermia; GSK-3β; β-catenin
21.  Xenon Preconditioning: The Role of Prosurvival Signaling, Mitochondrial Permeability Transition and Bioenergetics in Rats 
Anesthesia and analgesia  2009;108(3):858-866.
BACKGROUND
Similar to volatile anesthetics, the anesthetic noble gas xenon protects the heart from ischemia/reperfusion injury, but the mechanisms responsible for this phenomenon are not fully understood. We tested the hypothesis that xenon-induced cardioprotection is mediated by prosurvival signaling kinases that target mitochondria.
METHODS
Male Wistar rats instrumented for hemodynamic measurements were subjected to a 30 min left anterior descending coronary artery occlusion and 2 h reperfusion. Rats were randomly assigned to receive 70% nitrogen/30% oxygen (control) or three 5-min cycles of 70% xenon/30% oxygen interspersed with the oxygen/nitrogen mixture administered for 5 min followed by a 15 min memory period. Myocardial infarct size was measured using triphenyltetrazolium staining. Additional hearts from control and xenon-pretreated rats were excised for Western blotting of Akt and glycogen synthase kinase 3 β (GSK-3β) phosphorylation and isolation of mitochondria. Mitochondrial oxygen consumption before and after hypoxia/reoxygenation and mitochondrial permeability transition pore opening were determined.
RESULTS
Xenon significantly (P < 0.05) reduced myocardial infarct size compared with control (32 ± 4 and 59% ± 4% of the left ventricular area at risk; mean ± sd) and enhanced phosphorylation of Akt and GSK-3β. Xenon pretreatment preserved state 3 respiration of isolated mitochondria compared with the results obtained in the absence of the gas. The Ca2+ concentration required to induce mitochondrial membrane depolarization was larger in the presence compared with the absence of xenon pretreatment (78 ± 17 and 56 ± 17 μM, respectively). The phosphoinositol-3-kinase-kinase inhibitor wortmannin blocked the effect of xenon on infarct size and respiration.
CONCLUSIONS
These results indicate that xenon preconditioning reduces myocardial infarct size, phosphorylates Akt, and GSK-3β, preserves mitochondrial function, and inhibits Ca2+-induced mitochondrial permeability transition pore opening. These data suggest that xenon-induced cardioprotection occurs because of activation of prosurvival signaling that targets mitochondria and renders them less vulnerable to ischemia-reperfusion injury.
doi:10.1213/ane.0b013e318192a520
PMCID: PMC2748301  PMID: 19224794
22.  Susceptibility to myocardial ischemia reperfusion injury at early stage of type 1 diabetes in rats 
Background
Large body of evidences accumulated in clinical and epidemiological studies indicate that hearts of diabetic subjects are more sensitive to ischemia reperfusion injury (IRI), which results in a higher rate of mortality at post-operation than that of non-diabetes. However, experimental results are equivocal and point to either increased or decreased susceptibility of the diabetic hearts to IRI, especially at the early stage of the disease. The present study was designed to test the hypothesis that the duration/severity of the indexed ischemia is a major determinant of the vulnerability to myocardial IRI at early stage of diabetes.
Methods
Four weeks streptozotocin (STZ)-induced diabetic (D) and non-diabetic (C) Sprague–Dawley rats were randomly assigned to receive 30 or 45 min of left anterior descending artery ligation followed by 2 or 3 hours of reperfusion, respectively. Cardiac function was recorded by using Pressure-Volume (PV) conduction system. Myocardial infarct size was determined with triphenyltetrazolium chloride staining. Plasma Creatine kinase-MB (CK-MB), Lactate dehydrogenase (LDH) release, myocardial nitric oxide(NO) content and nitrotyrosine formation, 15-F2t-Isoprostane and plasma superoxide dismutase (SOD) were measured with colorimetric assays. Cardiomyocyte apoptosis was assessed by TUNEL staining. Myocardial TNFα, Caspase-3, STAT3, Akt, and GSK-3β were determined by Western blotting.
Results
Prolongation of ischemia but not reperfusion from 30 min to 45 min significantly increased infarct size in D compared to C rats (P < 0.05), accompanied with significantly increased plasma CK-MB (P < 0.05). Prolongation of the duration of either ischemia or reperfusion significantly increased plasma LDH release and myocardial 15-F2t-Isoprostane and reduced plasma SOD activity, with concomitant reduction of myocardial NO and increase of nitrotyrosine formation in D relative to C (P < 0.05). Prolongation of ischemia and reperfusion significantly reduced left ventricular ejection fraction and increased the peak rate of pressure, accompanied with increased end systolic pressure in D relative to C rats (P < 0.05) but reduced phosphorylations of myocardial STAT3 at site Ser727 and Akt at site Ser473 as well as GSK-3β at Ser 9 (P < 0.05).
Conclusions
Diabetic hearts, even at early stage of the disease are more sensitive to IRI, and this increased severity of post-ischemic myocardial injury depends more on the duration of ischemia than that of reperfusion.
doi:10.1186/1475-2840-12-133
PMCID: PMC3847499  PMID: 24041262
Ischemia reperfusion injury; Diabetes mellitus; Infarct size
23.  Inhibition of nuclear factor-κB by 6-O-acetyl shanzhiside methyl ester protects brain against injury in a rat model of ischemia and reperfusion 
Background
Recent studies have demonstrated an inflammatory response associated with the pathophysiology of cerebral ischemia. The beneficial effects of anti-inflammatory drugs in cerebral ischemia have been documented. When screening natural compounds for drug candidates in this category, we isolated 6-O-acetyl shanzhiside methyl ester (ND02), an iridoid glucoside compound, from the leaves of Lamiophlomis rotata (Benth.) Kudo. The objectives of this study were to determine the effects of ND02 on a cultured neuronal cell line, SH-SY5Y, in vitro, and on experimental ischemic stroke in vivo.
Methods
For TNF-α-stimulated SH-SY5Y cell line experiments in vitro, SH-SY5Y cells were pre-incubated with ND02 (20 μM or 40 μM) for 30 min and then incubated with TNF-α (20 ng/ml) for 15 min. For in vivo experiments, rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion for 23 h.
Results
ND02 treatment of SH-SY5Y cell lines blocked TNF-α-induced nuclear factor-κB (NF-κB) and IκB-α phosphorylation and increased Akt phosphorylation. LY294002 blocked TNF-α-induced phosphorylation of Akt and reduced the phosphorylation of both IκB-α and NF-κB. At doses higher than 10 mg/kg, ND02 had a significant neuroprotective effect in rats with cerebral ischemia and reperfusion (I/R). ND02 (25 mg/kg) demonstrated significant neuroprotective activity even after delayed administration 1 h, 3 h and 5 h after I/R. ND02, 25 mg/kg, attenuated histopathological damage, decreased cerebral Evans blue extravasation, inhibited NF-κB activation, and enhanced Akt phosphorylation.
Conclusion
These data show that ND02 protects brain against I/R injury with a favorable therapeutic time-window by alleviating cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and that these protective effects may be due to blocking of neuronal inflammatory cascades through an Akt-dependent NF-κB signaling pathway.
doi:10.1186/1742-2094-7-55
PMCID: PMC2946287  PMID: 20836895
24.  COMBINED LITHIUM AND VALPROATE TREATMENT DELAYS DISEASE ONSET, REDUCES NEUROLOGICAL DEFICITS AND PROLONGS SURVIVAL IN AN ALS MOUSE MODEL 
Neuroscience  2008;155(3):567-572.
Lithium and valproic acid (VPA) are two primary drugs used to treat bipolar disorder, and have been shown to have neuroprotective properties in vivo and in vitro. A recent study demonstrated that combined treatment with lithium and VPA elicits synergistic neuroprotective effects against glutamate excitotoxicity in cultured brain neurons, and the synergy involves potentiated inhibition of glycogen synthase kinase-3 (GSK-3) activity through enhanced GSK-3 serine phosphorylation (Leng et al., J Neurosci 28: 2576–2588, 2008). We therefore investigated the effects of lithium and VPA cotreatment on the disease symptom onset, survival time and neurological deficits in cooper zinc superoxide dismutase (SOD-1) G93A mutant mice, a commonly used mouse model of amyotrophic lateral sclerosis (ALS). The G93A ALS mice received twice daily intraperitoneal injections with LiCl (60 mg/kg), VPA (300 mg/kg) or lithium plus VPA, starting from the 30th day after birth and continuing until death. We found that combined treatment with lithium and VPA produced a greater and more consistent effect in delaying the onset of disease symptoms, prolonging the life span and decreasing the neurological deficit scores, compared with the results of monotreatment with lithium or VPA. Moreover, lithium in conjunction with VPA was more effective than lithium or VPA alone in enhancing the immunostaining of phospho-GSK-3βSer9 in brain and lumbar spinal cord sections. To our knowledge, this is the first demonstration of enhanced neuroprotection by a combinatorial approach using mood stabilizers in a mouse ALS model. Our results suggest that clinical trials using lithium and VPA in combination for ALS patients are a rational strategy.
doi:10.1016/j.neuroscience.2008.06.040
PMCID: PMC2709275  PMID: 18640245
lithium; valproic acid; GSK-3β; amyotrophic lateral sclerosis (ALS); G93A mice; behavioral deficits
25.  PPARγ Upregulation Induced by Vagus Nerve Stimulation Exerts Anti-Inflammatory Effect in Cerebral Ischemia/Reperfusion Rats 
Background
It is well known that peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated transcription factor, plays a protective role in anti-inflammatory responses in both acute and chronic central nerve system (CNS) insults. Emerging evidence in rats suggests that vagus nerve stimulation (VNS), while restraining inflammatory cytokine production in the peripheral nervous system, also exerts a significant CNS neuroprotective function against ischemic stroke injury. The aim of this study was to explore the role of PPARγ in VNS-mediated anti-inflammatory protection against ischemic stroke damage.
Material/Methods
Adult male Sprague-Dawley rats (total n=160) preconditioned through transfection with either PPARγ small interfering RNA (siRNA) or lentiviral vector without siRNA and surgically subjected to middle cerebral artery occlusion and reperfusion subsequently received VNS treatment at 30 min post-occlusion. The expression of PPARγ after VNS treatment was measured by real-time PCR and Western blotting, also supported by immunofluorescence staining. Subsequently, the neurological deficits scores, the infarct volume, and the brain histopathology were all evaluated. Additionally, the influence on the pro-inflammatory cytokines expression and neuro-immune cells activation was determined by ELISA and immunofluorescence staining.
Results
We found that VNS upregulated expression of PPARγ in ischemia penumbra, diminished the extent of ischemic infarct, alleviated neuronal injury, and suppressed pro-inflammatory cytokine expression and immune cell activation (P<0.05). However, rats with PPARγ silencing failed to manifest significant neuroprotection and anti-inflammatory effect induced by VNS treatment (p>0.05).
Conclusions
PPARγ may participate in the process by which VNS modulates the neuro-inflammatory response following ischemia/reperfusion in rats.
doi:10.12659/MSM.891407
PMCID: PMC4310716  PMID: 25619160
Cholinergic Fibers; Hypoxia-Ischemia, Brain; Peroxisome Proliferator-Activated Receptors; Vagus Nerve Stimulation

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