Simulated Reperfusion Research Articles

Direct neuronal protection by the protease-activated receptor PAR4 antagonist ML354 after experimental stroke in mice.

Thrombo-inflammation is a key feature of stroke pathophysiology and provides multiple candidate drug targets. Thrombin exerts coagulation-independent actions via protease-activated receptors (PAR), of which PAR1 has been implicated in stroke-associated neuroinflammation. The role of PAR4 in this context is less clear. This study examined if the selective PAR4 antagonist ML354 provides neuroprotection in experimental stroke and explored the underlying mechanisms. Mouse primary cortical neurons were exposed to oxygen-glucose deprivation (OGD) and simulated reperfusion ± ML354. For comparison, functional Ca2+-imaging was performed upon acute stimulation with a PAR4 activating peptide or glutamate. Male mice underwent sham operation or transient middle cerebral artery occlusion (tMCAO), with ML354 or vehicle treatment beginning at recanalization. A subset of mice received a platelet-depleting antibody. Stroke size and functional outcomes were assessed. Abundance of target genes, proteins, and cell markers was determined in cultured cells and tissues by qPCR, immunoblotting, and immunofluorescence. Stroke up-regulated PAR4 expression in cortical neurons in vitro and in vivo. OGD augments spontaneous and PAR4-mediated neuronal activity; ML354 suppresses OGD-induced neuronal excitotoxicity and apoptosis. ML354 applied in vivo after tMCAO reduced infarct size, apoptotic markers, macrophage accumulation, and interleukin-1β expression. Platelet depletion did not affect infarct size in mice with tMCAO ± ML354. Selective PAR4 inhibition during reperfusion improves infarct size and neurological function after experimental stroke by blunting neuronal excitability, apoptosis, and local inflammation. PAR4 antagonists may provide additional neuroprotective benefits in patients with acute stroke beyond their canonical antiplatelet action.

Cyclic AMP but Not Calmodulin as a Potential Wasoconstrictor in Simulated Reperfusion.

The phenomena of ischemia and reperfusion are associated with the pathological background of cardiovascular diseases. Ischemia is initiated by ischemia reperfusion injury (IRI), which involves disruption of intracellular signaling pathways and causes cell death. The aim of this study was to assess the reactivity of vascular smooth muscle cells in the conditions of induced ischemia and reperfusion, and to determine the mechanisms leading to contractility disorders. This study was conducted using classical pharmacometric methods on an isolated model of the rat caudal artery. The experiment consisted of the analysis of the final and initial perfusate pressure measurements after induction of arterial contraction with phenylephrine in the presence of forskolin and A7 hydrochloride, two ligands modifying the contractility of vascular smooth muscle cells (VSMC). The pharmacometric analysis showed that in simulated reperfusion, cyclic nucleotides have a vasoconstrictive effect, and calmodulin has a vasodilating effect. The responsiveness of vascular smooth muscle cells to the vasopressor effects of α1-adrenomimetics during reperfusion may change uncontrollably, and the effects of secondary messengers may be counter physiological. Further studies are needed to evaluate the function of other second messengers on VSMCs in the process of ischemia and reperfusion.

Forty-Eight Hour Ex Vivo Perfusion and Two-Hour Simulated Reperfusion after a Major Traumatic Upper Extremity Amputation

Abstract Background Extremity replantation as well as allotransplantation aim to restore form and function of the amputated limb. Both approaches, however, are clearly limited by the ischemic time. Ex vivo perfusion (EVP), already well-established in the field of solid organ transplantation, represents a promising tool to overcome this restriction. Methods We have currently established the technical requirements to provide EVP to severed limbs in a clinical hospital setting and hereby report of a 48-hour hypothermic EVP (EVP48) of an upper extremity amputated at the level of the proximal humerus, followed by a 2-hour simulated reperfusion (2SR) with donor blood. Results Muscle biopsies revealed histopathologically well preserved, vital muscle tissue after EVP24, and partially grouped muscle fiber necrosis with predominantly vital muscle tissue after EVP48 and 2SR. Analyses of perfusate samples showed a marked decline of biochemical muscle damage markers during EVP48. Cytokine analysis disclosed an isolated increase of the proinflammatory cytokines, interleukine-6, monocyte chemotactic protein-1, and interferon-γ, during EVP and 2SR. Magnetic resonance imaging, performed after EVP48, indicated partial muscle necrosis of the intrinsic hand muscles only, while no signs for infection or inflammation were present. Conclusion Our single case experience shows the general feasibility of an amputated limb ex vivo salvage perfusion setting to allow for delayed replantation up to 24 hours. Nevertheless, an accurate prior planning is crucial to ensure successful implementation of EVP in the acute clinical setting.

Abstract 14720: Marked Increase in Mitochondria Degradation in Engineered Heart Tissue During Reperfusion

Introduction: Engineered heart tissue (EHT) consisting of beating cardiomyocytes differentiated from human inducible pluripotent stem cells (hiPSCs) has the potential to recapitulate molecular and functional features of the human heart. Cardiac homeostasis is dependent on lysosomal removal of damaged mitochondria through selective autophagy (mitophagy). The level of mitophagy and cardioprotective impact during ischemia and reperfusion remains elusive and demands further investigation. Objective: To assess mitophagy during ischemia-reperfusion simulation in human EHTs. Methods and results: A hiPSC line was genetically modified using CRISPR/Cas9 knock-in to insert a fluorescent tag (mCherry or EGFP) on Translocase of the Outer Mitochondrial Membrane 20 ( TOMM20) gene resulting in hiPSCs with fluorescent mitochondria. The hiPSCs were differentiated to cardiomyocytes that were embedded in a fibrin hydrogel between two flexible silicone posts to generate EHTs. The EHTs (21 days or older) were subjected to 90 min of ischemia simulation followed by 120 min of simulated reperfusion with and without the lysosomal inhibitor Bafilomycin A1 (BafA1) to measure flux. Time-matched control EHTs remained in normal conditions (with and without BafA1). The EHTs were fixed and sections were immunostained with the lysosomal marker LAMP1A and analyzed by confocal microscopy. Co-localization of fluorescent mitochondria and stained lysosomes was observed after 120 min of reperfusion. To further assess this co-localization we used proximity ligation assay (PLA), an antibody-based assay allowing verification of close proximity (within 40 nm) of two proteins resulting in a single fluorescent signal (PLA dot). We performed the assay using anti-TOMM20 and anti-LAMP1A antibodies to localize mitochondrial content within lysosomes. Notably, there was a significant increase in the number of PLA dots in response to 120 min of reperfusion in the presence of BafA1 (3.41±0.56 fold vs. control, p<0.01; n=4), indicative of induced mitophagy flux. Conclusion: We observed a marked increase in lysosomal degradation of mitochondria in EHTs during reperfusion. The functional impact on EHTs during ischemia and reperfusion needs further research.

Natural Sources, Pharmacological Properties, and Health Benefits of Daucosterol: Versatility of Actions

Daucosterol is a saponin present in various natural sources, including medicinal plant families. This secondary metabolite is produced at different contents depending on species, extraction techniques, and plant parts used. Currently, daucosterol has been tested and explored for its various biological activities. The results reveal potential pharmacological properties such as antioxidant, antidiabetic, hypolipidemic, anti-inflammatory, immunomodulatory, neuroprotective, and anticancer. Indeed, daucosterol possesses important anticancer effects in many signaling pathways, such as an increase in pro-apoptotic proteins Bax and Bcl2, a decrease in the Bcl-2/Bax ratio, upregulation of the phosphatase and tensin homolog (PTEN) gene, inhibition of the PI3K/Akt pathway, and distortion of cell-cycle progression and tumor cell evolution. Its neuroprotective effect is via decreased caspase-3 activation in neurons and during simulated reperfusion (OGD/R), increased IGF1 protein expression (decreasing the downregulation of p-AKT3 and p-GSK-3b4), and activation of the AKT5 signaling pathway. At the same time, daucosterol inhibits key glucose metabolism enzymes to keep blood sugar levels within normal ranges. Therefore, this review describes the principal research on the pharmacological activities of daucosterol and the mechanisms of action underlying some of these effects. Moreover, further investigation of pharmacodynamics, pharmacokinetics, and toxicology are suggested.

Expression of atrial‑fetal light chains in cultured human cardiomyocytes after chemical ischemia‑reperfusion injury.

Atrial light chains (ALC1) are naturally present in adult heart atria, while ventricular light chains (VLC1) are predominant in ventricles. Degradation of VLC1 and re-expression of ALC1 in heart ventricles are associated with heart disorders in response to pressure overload. The aim of the current study was to investigate changes in myosin light chain expression after simulated ischemia and simulated reperfusion (sI/sR). Human cardiomyocytes (HCM) isolated from adult heart ventricles were subjected to chemical ischemia. The control group was maintained under aerobic conditions. Myocyte injury was determined by testing lactate dehydrogenase (LDH) activity. The gene expression of ALC1, VLC1 and MMP-2 were assessed by reverse transcription-quatitive PCR. Additionally, protein synthesis was measured using ELISA kits and MMP-2 activity was measured by zymography. The results revealed that LDH activity was increased in sI/sR cell-conditioned medium (P=0.02), confirming the ischemic damage of HCM. ALC1 gene expression and content in HCM were also increased in the sI/sR group (P=0.03 and P<0.001, respectively), while VLC1 gene expression after sI/sR was decreased (P=0.008). Furthermore, MMP-2 gene expression and synthesis were lower in the sI/sR group when compared with the aerobic control group (P<0.001 and P=0.03, respectively). MMP-2 activity was also increased in sI/sR cell-conditioned medium (P=0.006). In conclusion, sI/sR treatment led to increased ALC1 and decreased VLC1 expression in ventricular cardiomyocytes, which may constitute an adaptive mechanism to altered conditions and contribute to the improvement of heart function.

Cytoprotection of rat hepatocytes by desipramine in a model of simulated ischemia/reperfusion

We investigated the cytoprotective effect of desipramine (DMI) during in vitro simulated ischemia/reperfusion (I/R) of rat hepatocytes. Primary hepatocytes isolated from male Sprague-Dawley rats were subjected to 4 h of anoxia at pH 6.2 followed by normoxia at pH 7.4 for 2 h to simulate ischemia and reperfusion, respectively. During simulated reperfusion, some hepatocytes were reoxygenated using media containing 5 μM DMI. Necrotic cell death and the onset of mitochondrial permeability transition (MPT) were assessed using fluorometry and confocal microscopy. Changes in autophagic flux and autophagy-related proteins (ATGs) were analyzed by immunoblotting. DMI was shown to substantially delay MPT onset and suppress I/R related cell damage. Mechanistically, DMI treatment during reperfusion increased the expression level of the microtubule-associated protein 1A/1B-light chain 3 (LC3) processing enzymes, ATG4B and ATG7. Genetic knockdown of ATG4B abolished the cytoprotective effect of DMI. Together, these results indicate that DMI is a unique agent which enhances LC3 processing in an ATG4B-dependent way.

Role of AMPK in the protective effects exerted by triiodothyronine in ischemic-reperfused myocardium.

Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular 'fuel-gauge' enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3β phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.

The Effects of Targeted Temperature Management on Oxygen-Glucose Deprivation/Reperfusion-Induced Injury and DAMP Release in Murine Primary Cardiomyocytes

Introduction. Ischemia/Reperfusion (I/R) is a primary cause of myocardial injury after acute myocardial infarction resulting in the release of damage-associated molecular patterns (DAMPs), which can induce a sterile inflammatory response in the myocardial penumbra. Targeted temperature management (TTM) after I/R has been established for neuroprotection, but the cardioprotective effect remains to be elucidated. Therefore, we investigated the effect of TTM on cell viability, immune response, and DAMP release during oxygen-glucose deprivation/reperfusion (OGD/R) in murine primary cardiomyocytes. Methods. Primary cardiomyocytes from P1-3 mice were exposed to 2, 4, or 6 hours OGD (0.2% oxygen in medium without glucose and serum) followed by 6, 12, or 24 hours simulated reperfusion (21% oxygen in complete medium). TTM at 33.5°C was initiated intra-OGD, and a control group was maintained at 37°C normoxia. Necrosis was assessed by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation. OGD-induced DAMP secretions were assessed by Western blotting. Inducible nitric oxide synthase (iNOS), cytokines, and antiapoptotic RBM3 and CIRBP gene expressions were measured by quantitative polymerase chain reaction. Results. Increasing duration of OGD resulted in a transition from apoptotic programmed cell death to necrosis, as observed by decreasing caspase-3 cleavage and increasing LDH release. DAMP release and iNOS expression correlated with increasing necrosis and were effectively attenuated by TTM initiated during OGD. Moreover, TTM induced expression of antiapoptotic RBM3 and CIRBP. Conclusion. TTM protects the myocardium by attenuating cardiomyocyte necrosis induced by OGD and caspase-3 activation, possibly via induction of antiapoptotic RBM3 and CIRBP expressions, during reperfusion. OGD induces increased Hsp70 and CIRBP releases, but HMGB-1 is the dominant mediator of inflammation secreted by cardiomyocytes after prolonged exposure. TTM has the potential to attenuate DAMP release.

Aescin Protects Neuron from Ischemia-Reperfusion Injury via Regulating the PRAS40/mTOR Signaling Pathway.

Ischemic stroke is one of the major causes of disability; widely use of endovascular thrombectomy or intravenous thrombolysis leads to more attention on ischemia-reperfusion injury (I/R injury). Aescin, a natural compound isolated from the seed of the horse chestnut, has been demonstrated anti-inflammatory and antiedematous effects previously. This study was aimed at determining whether aescin could induce protective effects against ischemia-reperfusion injury and exploring the underlying mechanisms in vitro. Primary cultured neurons were subjected to 2 hours of oxygen-glucose deprivation (OGD) followed by 24 hours of simulated reperfusion. Aescin, which worked in a dose-dependent manner, could significantly attenuate neuronal death and reduce lactate dehydrogenase (LDH) release after OGD and simulated reperfusion. Aescin treatment at a concentration of 50 μg/ml provided protection with fewer side effects. Results showed that aescin upregulated the phosphorylation level of PRAS40 and proteins in the mTOR signaling pathway, including S6K and 4E-BP1. However, PRAS40 knockdown or rapamycin treatment was able to undermine and even abolish the protective effects of aescin; meanwhile, the levels of phosphorylation PRAS40 and proteins in the mTOR signaling pathway were obviously decreased. Hence, our study demonstrated that aescin provided neuronal protective effects against I/R injury through the PRAS40/mTOR signaling pathway in vitro. These results might contribute to the potential clinical application of aescin and provide a therapeutic target on subsequent cerebral I/R injury.

The small RNA microRNA-212 regulates sirtuin 2 expression in a cellular model of oxygen-glucose deprivation.

MicroRNA-212 has been found to play an important role in several types of diseases, but the functional and potential mechanisms of microRNA-212 in ischemic brain injury are still unclear. The aims of this study were to investigate the potential role of microRNA-212 in ischemic brain injury and to reveal potential molecular mechanisms. The rat oxygen-glucose deprivation and simulated reperfusion model was established to study the role of microRNA-212 in ischemic brain injury. The expression of microRNA-212 in oxygen-glucose deprivation and simulated reperfusion model and its effect on cell proliferation were measured by quantitative reverse transcription PCR and Cell Counting Kit-8 assay, respectively. The relationships between microRNA-212 and sirtuin 2 were confirmed by luciferase-reporter assay. We observed that microRNA-212 was downregulated after oxygen-glucose deprivation and simulated reperfusion treatment. Besides, the cells viabilities were increased/decreased in oxygen-glucose deprivation and simulated reperfusion model after transfection with microRNA-212 agomir (agonist of microRNA-212 action) and microRNA-212 antagomir (inhibitor of microRNA-212 action). In addition, luciferase and western blot experiments showed that microRNA-212 directly regulated sirtuin 2 changes. Furthermore, promotion of neuronal survival by microRNA-212 was blocked by overexpression of sirtuin 2, whereas the neuronal death induced by microRNA-212 inhibition was rescued by sirtuin 2 inhibition. Taken together, our study revealed that the role of miR-212 in the modulation of ischemic brain injury might be achieved by regulating sirtuin 2, which provides potential biomarkers and candidates for the treatment of cerebral ischemia.

The Association of Ascorbic Acid, Deferoxamine and N-Acetylcysteine Improves Cardiac Fibroblast Viability and Cellular Function Associated with Tissue Repair Damaged by Simulated Ischemia/Reperfusion.

Acute myocardial infarction is one of the leading causes of death worldwide and thus, an extensively studied disease. Nonetheless, the effects of ischemia/reperfusion injury elicited by oxidative stress on cardiac fibroblast function associated with tissue repair are not completely understood. Ascorbic acid, deferoxamine, and N-acetylcysteine (A/D/N) are antioxidants with known cardioprotective effects, but the potential beneficial effects of combining these antioxidants in the tissue repair properties of cardiac fibroblasts remain unknown. Thus, the aim of this study was to evaluate whether the pharmacological association of these antioxidants, at low concentrations, could confer protection to cardiac fibroblasts against simulated ischemia/reperfusion injury. To test this, neonatal rat cardiac fibroblasts were subjected to simulated ischemia/reperfusion in the presence or absence of A/D/N treatment added at the beginning of simulated reperfusion. Cell viability was assessed using trypan blue staining, and intracellular reactive oxygen species (ROS) production was assessed using a 2′,7′-dichlorofluorescin diacetate probe. Cell death was measured by flow cytometry using propidium iodide. Cell signaling mechanisms, differentiation into myofibroblasts and pro-collagen I production were determined by Western blot, whereas migration was evaluated using the wound healing assay. Our results show that A/D/N association using a low concentration of each antioxidant increased cardiac fibroblast viability, but that their separate administration did not provide protection. In addition, A/D/N association attenuated oxidative stress triggered by simulated ischemia/reperfusion, induced phosphorylation of pro-survival extracellular-signal-regulated kinases 1/2 (ERK1/2) and PKB (protein kinase B)/Akt, and decreased phosphorylation of the pro-apoptotic proteins p38- mitogen-activated protein kinase (p38-MAPK) and c-Jun-N-terminal kinase (JNK). Moreover, treatment with A/D/N also reduced reperfusion-induced apoptosis, evidenced by a decrease in the sub-G1 population, lower fragmentation of pro-caspases 9 and 3, as well as increased B-cell lymphoma-extra large protein (Bcl-xL)/Bcl-2-associated X protein (Bax) ratio. Furthermore, simulated ischemia/reperfusion abolished serum-induced migration, TGF-β1 (transforming growth factor beta 1)-mediated cardiac fibroblast-to-cardiac myofibroblast differentiation, and angiotensin II-induced pro-collagen I synthesis, but these effects were prevented by treatment with A/D/N. In conclusion, this is the first study where a pharmacological combination of A/D/N, at low concentrations, protected cardiac fibroblast viability and function after simulated ischemia/reperfusion, and thereby represents a novel therapeutic approach for cardioprotection.

Effects of Atorvastatin on Transient Sodium Currents in Rat Normal, Simulated Ischemia, and Reperfusion Ventricular Myocytes

Objectives: (1) To detect the whether the effects of simulated ischemia on I_Na of rat left ventricular myocytes in a time-dependent manner and the effects of atorvastatin on ischemia I_Na; (2) To investigate the effects of atorvastatin on I_Na of rat-simulated ischemia/reperfusion (I/R) ventricular cells. Materials and Methods: Ventricular cells were enzymatically isolated by Langendorff perfusion system. Whole-cell patch clamp was applied to detect I_Na level. Some elements of extracellular fluid were hanged to simulate the status of normal, I and R condition. Then the effects of atorvastatin on I_Na were observed. Results: (1) During simulated reperfusion, I_Na decreased and atorvastatin further suppressed the reduction degree. (2) At test potential –40 mV, no difference was detected among peak I_Na amplitude of ischemia for 20 min, reperfusion phase 3/5/7/9 min in continuous ischemia (I) group (p = 0.275). In I/R group, peak I_Na amplitude continuously decreased at 3 min (p = 0.005) and 9 min (p = 0.041). In atorvastatin intervention + I/R (Statin + I/R) group, peak I_Na amplitude at reperfusion 3 min decreased compared with ischemia phase (p = 0.000), while no significant difference was detected between 3 and 9 min (p = 0.858). The differences were significant at the same time point between groups. At reperfusion 3/5/7/9 min, peak I_Na of the I/R group was lower than the ischemia group (all p = 0.000), same as the Statin + I/R group (p = 0.000, p = 0.003, p = 0.006, and p = 0.001). Peak I_Na of the Statin + I/R group was higher than the I/R group at the same time point (p = 0.011, p = 0.033, p = 0.003, p = 0.003). There was no change in the I group during reperfusion phase (p > 0.05). In I/R group, V_1/2 (mV) shifted from –58.87 ± 3.36 to –54.33 ± 2.40, k (mV) shifted from 1.25 ± 0.59 to 1.91 ± 0.84 (p < 0.05). In the Statin + I/R group, V_1/2 (mV) increased from –57.80 ± 2.97 to –52.76 ± 3.14 (p < 0.01), no change was observed in k (p > 0.05). Conclusions: (1) In the status of reperfusion, I_Na decreased more than that in the status of ischemia. (2) Atorvastatin protected the cells from reduction of I_Na during long-time simulated (>15 min) I/R. (3) Overall, atorvastatin affected I_Na of the normal, simulated ischemic/reperfusion cell in rat left ventricle by blocking sodium channel ­directly.

Attenuating oxygen-glucose deprivation-caused autophagosome accumulation may be involved in sevoflurane postconditioning-induced protection in human neuron-like cells

Application of the commonly used volatile anesthetic sevoflurane after brain ischemia (sevoflurane postconditioning) attenuates ischemic brain injury. It is not known whether autophagy plays a role in this sevoflurane postconditioning-induced neuroprotection. Human SH-SY5Y cells were induced to become neuron-like cells. These cells were subjected to 1 h oxygen-glucose deprivation (OGD) and then exposed to sevoflurane for 1 h. Chloroquine, an inhibitor of autolysosomes, rapamycin, an autophagy inducer, or 3-methyladenine (3-MA), an autophagy inhibitor, were incubated with cells during OGD and sevoflurane exposure. OGD and the subsequent simulated reperfusion increased lactate dehydrogenase (LDH) release from the cells. This increase was dose-dependent inhibited by sevoflurane postconditioning. OGD increased the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3I and the expression of beclin-1 and p62. These increases were attenuated by sevoflurane. Sevoflurane alone did not have any effects on the expression of p62, beclin-1 and the ratio of LC3II to LC3I. Sevoflurane also enhanced the co-location of autophagosomes and lysosomes. Chloroquine increased the ratio of LC3II to LC3I, p62 and LDH release in cells subjected to OGD. Sevoflurane postconditioning attenuated OGD-induced inactivation of Akt and mechanistic target of rapamycin (mTOR). Inducing autophagosome generation by rapamycin attenuated sevoflurane postconditioning-reduced LDH release. Inhibition of autophagosome generation by 3-MA decreased OGD-induced LDH release. These results suggest that OGD increase autophagosome accumulation via increased formation of autophagosomes and reduced autophagosome clearance and that attenuation of OGD-induced autophagosome accumulation may contribute to sevoflurane postconditioning-induced cell protection.

LncRNA FGD5-AS1 acts as a competing endogenous RNA for miRNA-223 to lessen oxygen-glucose deprivation and simulated reperfusion (OGD/R)-induced neurons injury.

The purpose of this study was to evaluate whether FGD5-AS1 participates in oxygen-glucose deprivation and simulated reperfusion (OGD/R)-induced neurons injury and the detailed mechanism. An OGD/R model was established using the primary cortical neuron isolated from the brains of Sprague-Dawley rats. qRT-PCR and western blot were performed to de-tect the RNA and protein expression levels, respectively. Cell counting kit 8 (CCK8) and flow cytometry assays were used to evaluate the proliferation and apoptosis of neurons. The luciferase reporter assay was used to verify the interaction between lncRNA FGD5-AS1 and miRNA-223. We found that the expression of FGD5-AS1 is decreased in neurons suffering from OGD/R. Up-regulation of FGD5-AS1 could recover proliferation and inhibit apoptosis of OGD/R-injured neurons. In addition, the interaction between FGD5-AS1 and miRNA-223 were verified. The expression of miRNA-223 was negatively correlated with the level of FGD5-AS1. In turn, the expression of insulin-like growth factor-1 receptor (IGFIR, a target gene of miR-223) was positively associated with the level of FGD5-AS1. Simultaneously down-regulating miR-223 and over-expressing FGD5-AS1 as well as IGF1R exhibited an additional effect of extenuating OGD/R damage i.e. increasing neuron proliferation and reducing neuron apoptosis. In conclusion, our findings indicated that FGD5-AS1 may protect the neuron against OGD/R injury via acting as a ceRNA for miR-223 to mediate IGF1R expression, which contributes to a deeper understanding of ischemic stroke and provide a promising therapeutic target for this disease.

Impact of the hypoxic phenotype on the uptake and efflux of nanoparticles by human breast cancer cells

Breast cancer cells adapt to the hypoxic tumoral environment by undergoing changes in metabolism, cell signalling, endo-lysosomal receptor uptake and recycling. The resulting hypoxic cell phenotype has the potential to undermine the therapeutic efficacy of nanomedicines designed for endocytic uptake and specific intracellular trafficking. The aim of this study was to examine the impact of hypoxia and simulated reperfusion on the in vitro uptake and release of nanomedicines by human breast cancer cells. Cells were exposed to a hypoxic preconditioning treatment in 1% oxygen for 6 and 24 hours to induce temporal changes in the hypoxic circuit (e.g. HIF-1α expression). The preconditioned cells were then dosed with nanoparticles for 45 or 180 minutes emulating nanomedicine access following tumor reperfusion. Hypoxic preconditioning significantly increased nanoparticle retention by up to 10% when compared to normoxic cultures, with the greatest relative difference between normoxic and hypoxic cultures occurring with a 45 minute dosing interval. Exocytosis studies indicated that the preconditioned cells had a significantly increased nanoparticle efflux (up to 9%) when compared to normoxic cells. Overall, we were able to show that hypoxic preconditioning regulates both the endocytosis and exocytosis of nanomedicines in human breast cancer cells.

MiRNA-223 regulates ischemic neuronal injury by targeting the type 1 insulin-like growth factor receptor (IGF1R).

Cerebral ischemia injury seriously endangers human health and its molecular mechanism is still not fully understood. microRNA-223 (miR-223) has been reported to be involved in many physiological functions but the specific role of miRNA-223 in ischemic neuronal injury is still unclear. An oxygen-glucose deprivation and simulated reperfusion (OGD/R) model was constructed here to investigate the possible role miR-223 played in ischemic neuronal injury. The expression of miRNA-223 in the OGD/R model and its effect on cell proliferation were studied by qPCR and CCK8 assay. We observed that miR-223 was significantly over-expressed after OGD/R treatment and it suppressed significantly cortical neurons proliferation. To further study the mechanism involved, we predicted and examined the potential targets of miR-223 by targetscan, qPCR, western blot and luciferase reporter assay. We found that the expression level of type 1 insulin-like growth factor receptor (IGF1R) was negatively associated with the level of miR-223. Furthermore, the relative luciferase activity of pmirGLO-WT was inhibited obviously, while no significant change was observed in the pmirGLO-Mut group, indicating that miR-223 could bind to IGF1R. Similar cell proliferation suppression caused by miR-223 antagomir was observed when IGF1R was silenced. On the contrary, when cortical neurons were co-treated with miR-223 agomir and the cDNA of IGF1R which did not contain 3'- untranslated region, the inhibition caused by miR-223 disappeared. Our results suggested that miR-223 may suppress proliferation of cortical neurons that were treated with OGD/R via inhibiting IGF1R expression.

Scutellarin protects oxygen/glucose-deprived astrocytes and reduces focal cerebral ischemic injury.

Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxidative effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco's modified Eagle's medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen species in astrocytes subjected to ischemia-reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosin (3-NT) in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin improved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.

Peritransplant Energy Changes and Their Correlation to Outcome After Human Liver Transplantation.

The ongoing shortage of donor livers for transplantation and the increased use of marginal livers necessitate the development of accurate pretransplant tests of viability. Considering the importance energy status during transplantation, we aimed to correlate peritransplant energy cofactors to posttransplant outcome and subsequently model this in an ex vivo setting. Sequential biopsies were taken from 19 donor livers postpreservation, as well as 30 minutes after portal venous reperfusion and hepatic arterial reperfusion and analyzed by liquid chromatography-mass spectrometry for energetic cofactors (adenosine triphosphate [ATP]/adenosine diphosphate [ADP]/adenosine monophosphate [AMP], nicotinamide adenine dinucleotide /NAD, nicotinamide adenine dinucleotide phosphate / nicotinamide adenine dinucleotide phosphate , flavin adenine dinucleotide , glutathione disulfide/glutathione). Energy status was correlated to posttransplant outcome. In addition, 4 discarded human donation after circulatory death livers were subjected to ex vivo reperfusion, modeling reperfusion injury and were similarly analyzed for energetic cofactors. A rapid shift toward higher energy adenine nucleotides was observed following clinical reperfusion, with a 2.45-, 3.17- and 2.12-fold increase in ATP:ADP, ATP:AMP and energy charge after portal venous reperfusion, respectively. Seven of the 19 grafts developed early allograft dysfunction. Correlation with peritransplant cofactors revealed a significant difference in EC between early allograft dysfunction and normal functioning grafts (0.09 vs 0.31, P < 0.05). In the simulated reperfusion model, a similar trend in adenine nucleotide changes was observed. A preserved energy status appears critical in the peritransplant period. Levels of adenine nucleotides change rapidly after reperfusion and ratios of ATP/ADP/AMP after reperfusion are significantly correlated to graft function. Using these markers as a viability test in combination with ex vivo reperfusion may provide a useful predictor of outcome that incorporates donor, preservation, and reperfusion factors.

Comparison of normothermic and hypothermic perfusion in porcine kidneys donated after cardiac death

BackgroundNormothermic machine perfusion (NMP) is an alternative strategy for preserving kidneys donated after cardiac death (DCD). The relative efficacy of prolonged NMP compared to hypothermic machine perfusion (HMP) in DCD kidneys with moderate ischemic injury is undetermined. This study compares NMP and HMP kidney preservation in a porcine DCD model. MethodsTen porcine kidneys underwent NMP or HMP preservation following 45 minutes of warm ischemia and 5 hours of cold ischemia. After 8 hours of machine preservation, hemodynamic stability, renal function, perfusate biomarkers, and histologic integrity were assessed in a simulated reperfusion model. ResultsDuring simulated reperfusion, no differences were observed in oxygen consumption, urine production, creatinine clearance, fractional excretion of sodium, proteinuria, and perfusate levels of lactate dehydrogenase and aspartate aminotransferase. Resistance was no different after 30 minutes of simulated reperfusion. Histologically, NMP kidneys demonstrated increased vacuolization after preservation and greater loss of tubular integrity after simulated reperfusion. Perfusate levels of alkaline phosphatase (AP) and gamma glutamyltransferase (GGT) were higher in NMP kidneys during preservation, but upon simulated reperfusion, AP and GGT levels were higher in HMP-preserved kidneys. Peak AP and GGT during simulated reperfusion of HMP kidneys were over 14 times higher than peak AP and GGT during preservation of NMP kidneys. ConclusionsNMP provided comparable preservation of renal function as HMP and minimized AP and GGT release upon reperfusion.