Reoxygenation Research Articles

METTL3-deficiency Suppresses Neural Apoptosis to Induce Protective Effects in Cerebral I/R Injury via Inhibiting RNA m6A Modifications: A Pre-clinical and Pilot Study.

N6-Methyladenosine (m6A) RNA methylation involves in regulating the initiation, progression and aggravation of cerebral ischemia-reperfusion (I/R) injury, however, the detailed functions and mechanisms by which m6A drives cerebral I/R injury are not fully understood. This study found that methyltransferase-like 3 (METTL3) m6A-dependently regulated cerebral I/R injury trough regulating a novel LncRNA ABHD11-AS1/miR-1301-3p/HIF1AN/HIF-1α axis. Specifically, the middle cerebral artery occlusion (MCAO)/reperfusion mice models and glucose deprivation (OGD)/reoxygenation (RX) astrocyte cell models were respectively established, and we verified that METTL3, ABHD11-AS1 and HIF1AN were upregulated, whereas miR-1301-3p and HIF-1α were downregulated in both MCAO/reperfusion mice tissues and OGD/RX astrocytes. Mechanical experiments confirmed that METTL3 m6A dependently increased stability and expression levels of ABHD11-AS1, and elevated ABHD11-AS1 sponged miR-1301-3p to upregulate HIF1AN, resulting in the downregulation of HIF-1α. Moreover, silencing of METTL3 rescued MCAO/reperfusion and OGD/RX-induced oxidative stress-associated cell apoptosis and cell cycle arrest in both mice brain tissues in vivo and the mouse primary astrocytes in vitro, which were abrogated by overexpressing ABHD11-AS1 and downregulating miR-1301-3p. Taken together, our study firstly reported a novel METTL3/m6A/ ABHD11-AS1/miR-1301-3p/HIF1AN/HIF-1α signaling cascade in regulating the progression of cerebral I/R injury, and future work will focus on investigating whether the above genes can be used as biomarkers for the treatment of cerebral I/R injury by performing clinical studies.

Changes in nanomechanical properties of single neuroblastoma cells as a model for oxygen and glucose deprivation (OGD)

Although complex, the biological processes underlying ischemic stroke are better known than those related to biomechanical alterations of single cells. Mechanisms of biomechanical changes and their relations to the molecular processes are crucial for understanding the function and dysfunction of the brain. In our study, we applied atomic force microscopy (AFM) to quantify the alterations in biomechanical properties in neuroblastoma SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) and reoxygenation (RO). Obtained results reveal several characteristics. Cell viability remained at the same level, regardless of the OGD and RO conditions, but, in parallel, the metabolic activity of cells decreased with OGD duration. 24 h RO did not recover the metabolic activity fully. Cells subjected to OGD appeared softer than control cells. Cell softening was strongly present in cells after 1 h of OGD and with longer OGD duration, and in RO conditions, cells recovered their mechanical properties. Changes in the nanomechanical properties of cells were attributed to the remodelling of actin filaments, which was related to cofilin-based regulation and impaired metabolic activity of cells. The presented study shows the importance of nanomechanics in research on ischemic-related pathological processes such as stroke.

Glutamate-induced modulation in energy metabolism contributes to protection of rat cortical slices against ischemia-induced damage.

Glutamate excitotoxicity contributes to neurodegeneration during cerebral ischemia. Recent studies in the protective effect of glutamate against ischemia and hypoxia have shown the need for questioning the role of glutamate in energy metabolism during ischemia. Current study investigates the effect of glutamate on energy substrate metabolites such as alpha-ketoglutarate, lactate, and pyruvate release during control, oxygen-glucose deprivation (OGD), and reoxygenation (REO) conditions. The effects of 0.5 and 2 mM glutamate on spontaneous alpha-ketoglutarate, lactate, and pyruvate release were tested in vitro, on acute rat cortical slices. Alpha-ketoglutarate, lactate, and pyruvate levels were determined by HPLC with UV detector. We observed that glutamate added into medium significantly increased alpha-ketogluarate release under control conditions. Although OGD and REO also had a glutamate-like effect, only REO-induced rise further enhanced by glutamate. In contrast to alpha-ketoglutarate, both OGD and REO conditions caused significant declines in pyruvate and lactate outputs. While OGD and REO-induced declines in pyruvate outputs were further potentiated, lactate output was not altered by glutamate added into the medium. Glutamate and alpha-ketoglutarate, moreover, also ameliorated OGD- and REO-induced losses in 2,3,5-triphenyltetrazolium chloride staining with a similar degree. These results indicate that glutamate probably increases alpha-ketoglutarate production as an alternative energy source for use in the TCA cycle under energy-depleted conditions. Thus, increasing the alpha-ketoglutarate production may represent a new therapeutic intervention for neurodegenerative disorders, including cerebral ischemia.

Differential Regulation of mTOR Complexes with miR-302a Attenuates Myocardial Reperfusion Injury in Diabetes.

SummaryPersistent activation of mTOR (mammalian target of rapamycin) in diabetes increases the vulnerability of the heart to ischemia/reperfusion (I/R) injury. We show here that infusion of rapamycin (mTOR inhibitor) at reperfusion following ischemia reduced myocardial infarct size and apoptosis with restoration of cardiac function in type 1 diabetic rabbits. Likewise, treatment with rapamycin protected hyperglycemic human-pluripotent-stem-cells-derived cardiomyocytes (HG-hiPSC-CMs) following simulated ischemia (SI) and reoxygenation (RO). Phosphorylation of S6 (mTORC1 marker) was increased, whereas AKT phosphorylation (mTORC2 marker) and microRNA-302a were reduced with concomitant increase of its target, PTEN, following I/R injury in diabetic heart and HG-hiPSC-CMs. Rapamycin inhibited mTORC1 and PTEN, but augmented mTORC2 with restoration of miRNA-302a under diabetic conditions. Inhibition of miRNA-302a blocked mTORC2 and abolished rapamycin-induced protection against SI/RO injury in HG-hiPSC-CMs. We conclude that rapamycin attenuates reperfusion injury in diabetic heart through inhibition of PTEN and mTORC1 with restoration of miR-302a-mTORC2 signaling.

LncRNA MALAT1 silencing protects against cerebral ischemia-reperfusion injury through miR-145 to regulate AQP4

BackgroundThe present study aimed to verify whether long noncoding RNA (lncRNA) MALAT1 is involved in brain tissue damage induced by ischemia-reperfusion injury, and to explore the mechanism by which MALAT1 regulates aquaporin 4 (AQP4).MethodsIn this study, we established glucose deprivation (OGD)/reoxygenation (RX) astrocyte cell model and middle cerebral artery occlusion (MCAO)/reperfusion mouse model in vitro and in vivo. Then cell counting kit-8 assay, flow cytometry analysis, Triphenyltetrazolium chloride (TTC) staining, and western blotting were used to determine cell viability, cell apoptosis, cerebral infarction volume, and the abundance of AQP4, respectively.ResultsWe found that the level of MALAT1 was significantly upregulated in both the MCAO/reperfusion model and OGD/RX model. Knockdown of MALAT1 increased cell viability and reduced cell apoptosis in MA-C cells, while an AQP4 siRNA combined with a siRNA targeting MALAT1 could not enhance this effect. Further experiments showed that MALAT1 positively regulated AQP4 expression via miR-145. The MALAT1 siRNA did not alleviate the exacerbation of damage after miR-145 inhibitor action. However, an miR-145 inhibitor reversed the protection effects of MALAT1, indicating that MALAT1 silencing protects against cerebral ischemia-reperfusion injury through miR-145. TTC staining showed that the infracted area of whole brain was significantly attenuated in treated with sh-MALAT1 group in vivo.ConclusionTaken together, our study confirmed that MALAT1 promotes cerebral ischemia-reperfusion injury by affecting AQP4 expression through competitively binding miR-145, indicating that MALAT1 might be a new therapeutic target for treatment cerebral ischemic stroke.

Aging protects rat cortical slices against to oxygen-glucose deprivation induced damage

Objective: In present study, we aimed to clarify effect of aging on the susceptibility of brain tissue to neurodegeneration induced by ischemia.Methods: Damage induced by oxygen-glucose deprivation (OGD) followed by reoxygenation (REO) were compared in cortical slices prepared from young (3 months of age) and aged (22-24 months of age) male Sprague Dawley rats.Results: After incubation of the slices in an oxygen and glucose containing control condition, 2,3,5-triphenyl tetrazolium chloride (TTC) staining intensity was found significantly high in aged cortical slices. Although thirty minutes incubation of the slices in OGD medium followed by REO (OGD-REO) caused similar decline in TTC staining in young and aged cortical slices, staining intensity was still significantly higher in the slices prepared from aged animals. Thirty minutes of OGD-REO, on the other hand, also caused more increase in lactate dehydrogenase (LDH) leakage from young slices. While water contents of the slices were almost equal under control condition, it was significantly high in young cortical slices after OGD-REO incubations. In contrary to these findings, OGD and REO caused more increases in S100B output from aged rat cortical slices. S100B levels in brain regions including the cerebral cortex were also found higher in aged rats.Conclusion: All these results indicate that, cortical slices prepared from aged male rats are significantly less responsive to in vitro OGD-REO induced alterations. Since protein S100B outputs were almost doubled from aged cortical slices, a possible involvement of this enhanced S100B output seems to be likely.

Exacerbation of oxygen-glucose deprivation-induced blood-brain barrier disruption: potential pathogenic role of interleukin-9 in ischemic stroke.

Interleukin (IL)-9 exerts a variety of functions in autoimmune diseases. However, its role in ischemic brain injury remains unknown. The present study explored the biological effects of IL-9 in ischemic stroke (IS). We recruited 42 patients newly diagnosed with IS and 22 age- and sex-matched healthy controls. The expression levels of IL-9 and percentages of IL-9-producing T cells, including CD3+CD4+IL-9+ and CD3+CD8+IL-9+ cells, were determined in peripheral blood mononuclear cells (PBMCs) obtained from patients and control individuals. We also investigated the effects of IL-9 on the blood-brain barrier (BBB) following oxygen-glucose deprivation (OGD) and the potential downstream signaling pathways. We found that patients with IS had higher IL-9 expression levels and increased percentages of IL-9-producing T cells in their PBMCs. The percentages of CD3+CD4+IL-9+ and CD3+CD8+IL-9+ T cells were positively correlated with the severity of illness. In in vitro experiments using bEnd.3 cells, exogenously administered IL-9 exacerbated the loss of tight junction proteins (TJPs) in cells subjected to OGD plus reoxygenation (RO). This effect was mediated via activation of IL-9 receptors, which increased the level of endothelial nitric oxide synthase (eNOS), as well as through up-regulated phosphorylation of signal transducer and activator of transcription 1 and 3 and down-regulated phosphorylatedprotein kinase B/phosphorylated phosphatidylinositol 3-kinase signaling. These results indicate that IL-9 has a destructive effect on the BBB following OGD, at least in part by inducing eNOS production, and raise the possibility of targetting IL-9 for therapeutic intervention in IS.

Mechanisms Linking Chronic Obstructive Pulmonary Disease and Stroke

Recent studies have shown that chronic obstructive pulmonary disease (COPD) is associated with an increased stroke risk2 and mortality3. Cigarette smoke is a shared risk factor for COPD and stroke. Therefore it is conceivable that the link between COPD and stroke may relate, at least in part, to cigarette smoke. During stroke, excess reactive oxygen species (ROS) or oxidative stress disrupts the blood‐brain barrier (BBB), which in turn can accentuate brain injury. Importantly, cigarette smoke increases ROS production by endothelial cells. Therefore, we tested the hypothesis that COPD/smoking worsens stroke severity by exacerbating oxidative‐induced BBB disruption after stroke. Monolayers of mouse cerebral microvascular endothelial cells (bEnd.3 cells) were pre‐treated with either vehicle, 5, 10, 20 or 40% CSE (1 h) prior to exposure to oxygen glucose deprivation (OGD) (4 h) and reoxygenation (RO) (23 h). Cells cultured under normoxic conditions (27 h) were used as controls. We assessed BBB disruption by measuring paracellular permeability (FITC‐dextran), tight junction protein expression (Western blotting), and cell viability (MTS assay). Furthermore, we measured superoxide (L‐012‐enhanced chemiluminescence) and hydrogen peroxide (H2O2) production (Amplex Red fluorescence). 10% CSE increased the paracellular permeability of bEnd.3 cells cultured under normoxic conditions, indicating BBB disruption. Furthermore, 10% CSE exacerbated (~2‐fold) the hyperpermeability of bEnd.3 cells exposed to OGD + RO (10% CSE, 5±0.9 vs. vehicle, 2.8±0.7; fold‐change relative to normoxic controls, n=8, P<0.05), indicating a worsening of BBB disruption after ischaemic‐like conditions. However, this was found to be independent of increased tight junction protein degradation (occludin or ZO‐1) or cell death (n=6, P>0.05). Contrary to our hypothesis, CSE (10% or 20%) significantly attenuated OGD/RO‐induced elevations in superoxide production (10% CSE, 97±6.3, 20% CSE 75±5 vs. vehicle, 147±16; % normoxic, n=5, P<0.05), but had no significant effect on H2O2 production (n=5, P>0.05). Thus, CSE worsens BBB disruption in an in vitro model of stroke; however, this appears to be unrelated to increased oxidative stress. Although, much more research is needed, these tantalizing findings raise the possibility that such an effect may play a role in the link between COPD and stroke.Support or Funding InformationSupported by NHMRC funding

Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5% fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5% FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5% FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.

Disrupted intestinal structure in a rat model of intermittent hypoxia.

Obstructive sleep apnea (OSA) is a chronic condition characterized by chronic intermittent hypoxia (IH) and subsequent reoxygenation (ROX). The gastrointestinal system, which is particularly sensitive to tissue hypoxia and reduced perfusion, is likely to be affected by OSA. A rat model of IH was used to analyze oxidative stress-associated genes and tight junction proteins by reverse transcription‑quantitative polymerase chain reaction. Subsequently, altered morphology of the duodenal mucosa and elevated Chiu scores were observed in the IH‑exposed rats. In addition, IH exposure resulted in upregulation of the nicotinamide adenine dinucleotide phosphate(NADPH) oxidase subunits, NADPH oxidase2 and p22phox, in the small intestine, and upregulation of transcription factors, including hypoxia‑inducible factor-1, nuclear factor‑κB and activator protein-1. Furthermore, the mRNA expression levels of intestinal tight junction (TJ)-related proteins, claudin-1 and claudin-4, were decreased in the IH‑exposed group, as compared with in the control group. In conclusion, the present study demonstrated that OSA, which is characterized by IH and ROX, may lead to disruption of the duodenum. The mechanism underlying the effects of OSA on duodenal morphology may be associated with increased oxidative stress and activation of transcription factors, subsequently inducing intestinal TJ disruption and intestinal injury.

Abstract 16115: Rapamycin Protects Diabetic Heart Against Ischemia-reperfusion Injury by Inhibiting JNK and STAT4

Background: Persistent activation of the mammalian target of rapamycin (mTOR) is implicated in diabetic complications and plays a critical role in myocardial injury following ischemia/reperfusion. Recently we reported that mTOR inhibition with rapamycin improves cardiac function in type 2 diabetic (T2D) mice through attenuation of oxidative stress and alteration of antioxidants and contractile protein expression. Since increased JNK and STAT4 activities have been strongly associated with T2D-induced cardiovascular complications, we hypothesized that rapamycin would reduce myocardial infarction (MI) in T2D mice through inhibition of JNK and STAT4. Methods and Results: Adult male wild type (C57) or db/db T2D mice were treated daily for 28 days with rapamycin (0.25 mg/kg, i.p.). Following treatment, the hearts were subjected to global I (30 min) and R (60 min) in Langendorff mode. In addition, cardiomyocytes were isolated from treated mice and subjected to 40 min simulated ischemia (SI) and 1 hr or 18 hr reoxygenation (RO) to assess necrosis (by trypan blue staining) or apoptosis (by TUNEL assay), respectively. Diabetes exacerbated ischemic infarct size (measured by tetrazolium chloride staining) in db/db mice as compared to C57 mice (Fig. A), and also enhanced cardiomyocyte necrosis (Fig. B) and apoptosis (Fig. C). Rapamycin treatment reduced infarct size following I/R and protected adult cardiomyocytes against necrosis and apoptosis following SI/RO. Rapamycin also improved post-ischemic contractile function in db/db mice (Fig. D). Western blot analysis revealed a marked increase in phosphorylation of JNK and STAT4 in diabetic hearts, which were reduced in rapamycin-treated diabetic hearts (Fig. E). Conclusion: Rapamycin treatment reduces MI possibly by inhibiting JNK and STAT4 in T2D heart. We propose that inhibition of JNK and STAT4 with rapamycin therapy may be an attractive strategy to improve prognosis in diabetics following MI.

Sp3/REST/HDAC1/HDAC2 Complex Represses and Sp1/HIF-1/p300 Complex Activates ncx1 Gene Transcription, in Brain Ischemia and in Ischemic Brain Preconditioning, by Epigenetic Mechanism.

The Na(+)-Ca(2+) exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in ischemic brain preconditioning (PC) by hypoxia-inducible factor 1 (HIF-1). Because ncx1 brain promoter (ncx1-Br) has five putative consensus sequences, named Sp1A-E, for the specificity protein (Sp) family of transcription factors (Sp1-4), we investigated the role of this family in regulating ncx1 transcription in rat cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1-Br in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C-E. Furthermore, by transient middle cerebral artery occlusion (tMCAO) in rats, the transcriptional repressors Sp3 and REST colocalized with the two histone-deacetylases (HDACs) HDAC1 and HDAC2 on the ncx1-Br, with a consequent hypoacetylation. Contrarily, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalized with histone acetyltransferase p300 on ncx1-Br with a consequent hyperacetylation. In addition, in neurons silenced with siRNA of NCX1 and subjected to oxygen and glucose deprivation (OGD) (3 h) plus reoxygenation (RX) (24 h), the neuroprotection of Class I HDAC inhibitor MS-275 was counteracted, whereas in neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD/RX), the neurotoxic effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by the Sp3/REST/HDAC1/HDAC2 complex in tMCAO and by the Sp1/HIF-1/p300 complex in PC+tMCAO and that epigenetic intervention, by modulating the acetylation of ncx1-Br, may be a strategy for the development of innovative therapeutic intervention in stroke.

Altered fractionation outcomes for hypoxic head and neck cancer using theHYP-RTMonte Carlo model

Altered fractionation radiotherapy is simulated on a set of virtual tumours to assess the total doses required for tumour control compared with clinical head and neck data and the doses required to control hypoxic vs well-oxygenated tumours with different radiobiological properties. The HYP-RT model is utilised to explore the impact of tumour oxygenation and the onset times of accelerated repopulation (AR) and reoxygenation (ROx) during radiotherapy. A biological effective dose analysis is used to rank the schedules based on their relative normal tissue toxicities. Altering the onset times of AR and ROx has a large impact on the doses required to achieve tumour control. Immediate onset of ROx and 2-week onset time of AR produce results closely predicting average human outcomes in terms of the total prescription doses in clinical trials. Modifying oxygen enhancement ratio curves based on dose/fraction significantly reduces the dose (5-10 Gy) required for tumour control for hyperfractionated schedules. HYP-RT predicts 10×1.1 Gy per week to be most beneficial, whereas the conventional schedule is predicted as beneficial for early toxicity but has average-poor late toxicity. HYP-RT predicts that altered radiotherapy schedules increase the therapeutic ratio and may be used to make predictions about the prescription doses required to achieve tumour control for tumours with different oxygenation levels and treatment responses. Oxic and hypoxic tumours have large differences in total radiation dose requirements, affected by AR and ROx onset times by up to 15-25 Gy for the same fractionation schedule.

NCX1 is a new rest target gene: Role in cerebral ischemia

The Na+–Ca2+ exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasmamembrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2)In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3)By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4)Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5)REST knocking down, along with the deriving NCX1overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration.Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia.

Adenoviral transfer of PKGIα; attenuates apoptosis and necrosis in adipose derived stem cells

Adenoviral transfer of protein kinase G-Iα (PKGIα) protects myocytes from ischemia but the effect of PKGIα on adipose derived stem cells (ASCs) remains unknown. We hypothesize that PKGIα will attenuate necrosis and apoptosis of ASCs in an ischemic environment. ASCs were infected with adenoviral vectors containing PKGIα or a mutant then subjected to simulated ischemia (SI) and reoxygenation (RO) for necrosis or apoptosis studies. To determine the role of PKG overexpression in vivo, adult male ICR mice underwent permanent left coronary artery occlusion before receiving treatment of 2x10^5 cells overexpressing PKGIα or mutant. 2 weeks after surgery, transthoracic echocardiography (TTE) was performed to assess left ventricular (LV) function. PKGIα overexpression reduced necrosis as seen by a decrease in trypan blue positive cells and LDH release when compared to SI/RO control (Fig 1 A, B). TUNEL revealed that apoptosis was also significantly reduced in cells overexpressing PKGIα (5.1± 0.47%) as compared to SI/RO control (12.8±1.07%). ASCs infected with the inactive PKGIαK390A mutant lacked protection. TTE results showed reduced LV dilatation and preservation of ejection fraction with PKG overexpression (Fig 1 C, D). Therefore, adenoviral gene transfer of PKGIα may provide a novel strategy for protecting ASCs against ischemia which could translate to a favorable function in hearts following ischemia. Figure 1Open in figure viewerPowerPoint

Protein aggregation in neurons following OGD: a role for Na+ and Ca2+ ionic dysregulation

In this study, we investigated whether disruption of Na(+) and Ca(2+) homeostasis via activation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchange (NCX(rev)) affects protein aggregation and degradation following oxygen-glucose deprivation (OGD). Cultured cortical neurons were subjected to 2 h OGD and 1-24 h reoxygenation (REOX). Redistribution of ubiquitin and formation of ubiquitin-conjugated protein aggregates occurred in neurons as early as 2 h REOX. The protein aggregation progressed further by 8 h REOX. There was no significant recovery at 24 h REOX. Moreover, the proteasome activity in neurons was inhibited by 80-90% during 2-8 h REOX and recovered partially at 24 h REOX. Interestingly, pharmacological inhibition or genetic ablation of NKCC1 activity significantly decreased accumulation of ubiquitin-conjugated protein aggregates and improved proteasome activity. A similar protective effect was obtained by blocking NCX(rev) activity. Inhibition of NKCC1 activity also preserved intracellular ATP and Na(+) homeostasis during 0-24 h REOX. In a positive control study, disruption of endoplasmic reticulum Ca(2+) with thapsigargin triggered redistribution of free ubiquitin and protein aggregation. We conclude that overstimulation of NKCC1 and NCX(rev) following OGD/REOX partially contributes to protein aggregation and proteasome dysfunction as a result of ionic dysregulation.

Abstract 2894: Essential Role of ERK 1/2 in Sildenafil-Induced Early and Delayed Cardioprotection in Mice

Background: Sildenafil (SIL), a potent inhibitor of phosphodiesterase-5 induces powerful protection against myocardial ischemia-reperfusion (I-R) injury through activation of protein kinase G (PKG). However, the downstream targets of PKG in SIL-induced cardioprotection remain unclear. We hypothesized that PKG-dependent activation of survival kinase, ERK may play a critical role in SIL-induced cardioprotection in mice. Methods & Results: Ventricular myocytes were isolated from adult male ICR mice and exposed to 40 min of simulated ischemia (SI) with/without 1 hr pre-incubation of SIL (1 μM). Myocyte necrosis and apoptosis were determined after 1 hr or 18 hrs of reoxygenation (RO) using trypan blue or TUNEL assay, respectively. Pretreatment with SIL protected cardiomyocytes after SI-RO (necrosis 18.5±0.5% and apoptosis 6.6±0.7%; n=4, p<0.001) as compared with controls (necrosis 42.1±1.8% and apoptosis 23.3±0.9%). Co-incubation of PD98059 (20 μM), a selective ERK1/2 inhibitor blocked both anti-necrotic and anti-apoptotic protection in cardiomyocytes. Furthermore, intra-coronary infusion of SIL (1 μM) in Langendorff isolated mouse hearts 10 min prior to zero-flow global I (20 min) and R (30 min) significantly reduced myocardial infarct size (from 29.4±2.4% to 16.0±3.0%; p<0.05, n=6). Co-treatment of PD98059 abrogated SIL-induced protection (33.0±5.9; n=4). To evaluate the role of ERK1/2 in delayed cardioprotection, mice were treated with saline or SIL (0.7 mg/kg i.p.) 24 hours before global I-R in Langendorff mode. PD98059 (1 mg/kg) was administered (i.p.) 30 min before the treatment of SIL. Infarct size was reduced from 27.6±3.3% in saline-treated controls to 6.9±1.2% in SIL-treated mice (P<0.05, n=6). The delayed protective effect of SIL was also abolished by PD98059 (22.5±2.3%). Western Blots revealed that SIL significantly increased phosphorylation of ERK1/2 which was blocked by PKG inhibitor, KT5823 in the heart and adult myocytes. Selective knockdown of PKG in cardiomyocytes with short hairpin RNA of PKG also blocked the phosphorylation of ERK1/2. Conclusion: SIL-induced cardioprotection involves the activation and phosphorylation of ERK which appear to be intimately linked with a PKG-dependent survival pathway. This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).

A concerted role of Na+ -K+ -Cl- cotransporter and Na+/Ca2+ exchanger in ischemic damage.

Na+-K+-Cl(-) cotransporter isoform 1 (NKCC1) and Na+/Ca2+ exchanger isoform 1 (NCX1) were expressed in cortical neurons. Three hours of oxygen and glucose deprivation (OGD) significantly increased expression of full-length NCX1 protein ( approximately 116 kDa), which remained elevated during 1 to 21 h reoxygenation (REOX) and was accompanied with concurrent cleavage of NCX1. Na+/Ca2+ exchanger isoform 1 heterozygous (NCX1+/-) neurons with approximately 50% less of NCX1 protein exhibited approximately 64% reduction in NCX-mediated Ca2+ influx. Expression of NCX1 and NKCC1 proteins was reduced in double heterozygous (NCX1+/-/NKCC1+/-) neurons. NCX-mediated Ca2+ influx was nearly abolished in these neurons. Three-hour OGD and 21-h REOX caused approximately 80% mortality rate in NCX1+/+ neurons and in NCX1+/- neurons. In contrast, NKCC1+/- neurons exhibited approximately 45% less cell death. The lowest mortality rate was found in NCX1+/-/NKCC1+/- neurons ( approximately 65% less neuronal death). The increased tolerance to ischemic damage was also observed in NCX1+/-/NKCC1+/- brains after transient cerebral ischemia. NCX1+/-/NKCC1+/- mice had a significantly reduced infarct volume at 24 and 72 h reperfusion. In conclusion, these data suggest that NKCC1 in conjunction with NCX1 plays a role in reperfusion-induced brain injury after ischemia.

ERK1/2-p90RSK-mediated Phosphorylation of Na+/H+ Exchanger Isoform 1: A ROLE IN ISCHEMIC NEURONAL DEATH

The function and regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the V(max) for NHE1 but also shifted the K(m) toward decreased [H(+)](i). These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.

Neuroprotective effects of cactus polysaccharide on oxygen and glucose deprivation induced damage in rat brain slices.

1. The neuroprotective effect of cactus polysaccharide (CP) on oxygen and glucose deprivation (OGD) and reoxygenation (REO)-induced damage in the cortical and hippocampal slices of rat brain was investigated. 2. Cell viability was evaluated by using the 2, 3, 5-triphenyl tetrazolium chloride (TTC) method. The fluorescence of propidium iodide (PI) staining was used for quantification of cellular survival, and lactate dehydrogenase (LDH) activity in incubation medium was assessed by LDH assay to evaluate the degree of injury. 3. The OGD ischemic condition significantly decreased cellular viability and increased LDH release in the incubation medium. CP (0.2 mg/l approximately 2 mg/l) protected brain slices from OGD injury in a dosage dependent manner as demonstrated by increased A 490 value of TTC, decreased PI intensity and LDH release. At the above concentration, CP also prevented the increase of nitric oxide (NO) content and inducible nitric oxide synthase (iNOS) activity induced by OGD. 4. CP can protect the brain slices (cortical and hippocampus) against injury induced by OGD. Its neuroprotective effect may be partly mediated by the NO/iNOS system induced by OGD insult.