24-h Reperfusion Research Articles

Lithium alleviates blood-brain barrier breakdown after cerebral ischemia and reperfusion by upregulating endothelial Wnt/β-catenin signaling in mice

Although upregulation of endothelial Wnt/β-catenin signaling may be used to treat blood-brain barrier (BBB) breakdown caused by cerebral ischemia/reperfusion injury, no agents based on this mechanism are available clinically. Lithium, a medication used for treating bipolar mood disorders, upregulates Wnt/β-catenin signaling, but whether lithium alleviates BBB breakdown after ischemic stroke by upregulating endothelial Wnt/β-catenin signaling is unclear. Here, we evaluated the BBB-protective effect of lithium in adult mice with 1-h middle cerebral artery occlusion and 48-h reperfusion (MCAO/R) by determining neurological outcomes, BBB function and related molecular components. Furthermore, we assessed the effect and dependence of lithium on Wnt/β-catenin signaling in brain microvascular endothelial cells in cell culture and in mice with conditional endothelial knockout of Wnt7 co-receptor Gpr124. Our data show that lithium treatment (3 mmol/kg) significantly decreased infarct volume (34.1 ± 1.8% versus 58.3 ± 2.8% in vehicle controls, P < 0.0001) and improved neurological outcomes of mice following MCAO/R. Importantly, lithium significantly increased BBB integrity shown by reduction of Evans blue leakage (by 45.7%, P = 0.0064) and blood IgG extravasation (by 65.8%, P < 0.0001) into infarcted brain tissue. Mechanistically, lithium upregulated the activity of endothelial Wnt/β-catenin signaling in vivo and in vitro, increased the protein levels of tight junctions (Claudin-5 and ZO-1), and reduced MMP-9 expression. Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, indicating the protection of lithium on BBB was mainly dependent on the Gpr124-mediated endothelial Wnt/β-catenin signaling. Taken together, our findings indicate that lithium may serve as a therapeutic candidate for treating the BBB breakdown in the early stage of ischemic stroke following reperfusion therapy.

Can MitoTEMPO protect rat sciatic nerve against ischemia-reperfusion injury?

Abdominal ischemia-reperfusion (I/R) is known to cause both structural and functional damage to sciatic nerve which is related to the oxidative stress. We investigated the protective effects of mitochondria-targeted antioxidant (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) on ischemia-reperfusion-induced nerve damage by using the conduction velocity distribution (CVD) calculations from in vitro compound nerve action potential (CNAP) recordings from rat sciatic nerve. Adult male Wistar albino rats were divided into three groups. The IR and IR + MT groups had aortic cross-clamping for 1h followed by 2h reperfusion, while SHAM group had the same procedure without cross-clamping. IR + MT group received 0.7mg/kg/day MitoTEMPO injection for 28days before I/R, while other groups received vehicle alone. Ischemia-reperfusion resulted in a significant decrease (p < .05) in maximum depolarizations (mV), areas (mV.ms), and maximum and minimum upstroke velocities (mV/ms) of CNAPs, while injection of MitoTEMPO showed a complete protective effect on these impairments. The histograms for CVD showed that I/R blocked the contribution of fast-conducting fibers (> 60m/s). MitoTEMPO prevented that blockage and caused a shift in the CVD. Functional nerve damage caused by I/R can be prevented by MitoTEMPO, which can enter mitochondria, the main source of reactive oxygen species (ROS).

Recovery of Human Embryonic Stem Cells-Derived Neural Progenitors Exposed to Hypoxic-Ischemic-Reperfusion Injury by Indirect Exposure to Wharton’s Jelly Mesenchymal Stem Cells Through Phosphatidyl-inositol-3-Kinase Pathway

Increasing evidence suggests that mesenchymal stem cells(MSCs) have beneficial effects in hypoxic ischemic reperfusion injury, but the underlying mechanisms are unclear. Here, we first examined the effect of OGD reperfusion injury on the vulnerability of human NPs derived from human embryonic stem cells (hESCs) with regard to cell survival and oxidative stress. Cellular deregulation was assessed by measuring glutathione levels, basal calcium and intracellular calcium [Ca2+]i response under KCl stimulation, as well as the key parameters of proliferation, glial progenitor marker expression and migration. Next, the influence of WJ-MSCs in recovering these parameters was evaluated, and the role of Phosphatidyl-inositol-3-Kinase(PI3K) pathway in actuating the protective effect was assessed. OGD reperfusion injury induced significant increases in cell death, ROS generation, oxidative stress susceptibility and decreased glutathione levels in NPs, accompanied by rises in basal [Ca2+]i, KCl-induced [Ca2+]i, expression of K+ leak channel(TASK1), and declines in proliferation, migration potential and glial progenitor population. The introduction of WJ-MSCs(after 2h of reperfusion) through a non-contact method brought about significant improvement in all these cellular parameters as observed after 24hrs, and the PI3K pathway played an important role in the neuroprotection process. Presence of WJ-MSCs increased the expression of survival signals like phosphorylated Akt/Akt and PI3K in the OGD-reperfused NPs. Our data clearly demonstrate for the first time that soluble factors from WJ-MSCs can not only ameliorate survival, proliferation, migration and glial progenitor expression of OGD-reperfused NPs, but also regulate their intracellular Ca2+ response to KCl stimulation and expression of TASK1 through the PI3K pathway.

Abstract 14412: Activation of Autophagic Flux Maintains Mitochondrial Homeostasis During Cardiac Ischemia/reperfusion Injury

Background and Hypothesis: Reperfusion injury accounts for ~50% of myocardial infarct size, and clinically efficacious therapies are lacking. Histone deacetylase (HDAC) inhibition enhances cardiomyocyte autophagic activity, mitochondria biogenesis, and blunts ischemia/reperfusion (I/R) injury when given at the time of reperfusion. However, as HDAC inhibition has pleiotropic effects, we will test whether augmentation of autophagic flux using a specific autophagy-inducing peptide, Tat-Beclin (TB), is cardioprotective. Methods: 8-12-week-old, wild-type, C57BL6 mice were randomized into three groups: vehicle control, Tat-Scrambled (TS) peptide, or Tat-Beclin (TB) peptide. Each group was subjected to I/R surgery (45min ischemia, 24h reperfusion). Infarct size, systolic function, and mitochondrial dynamics were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were used to test for cardiomyocyte specificity. Conditional cardiomyocyte ATG7 knockout (ATG7 KO) mice and ATG7 knockdown by siRNA in NRVMs were used to evaluate the role of autophagy. Results: TB treatment at reperfusion reduced infarct size by 20.1±6.3% (n=23, p&lt;0.02) and improved systolic function. Increased autophagic flux and reduced reactive oxygen species (ROS) were observed in the infarct border zone. The cardioprotective effects of TB were abolished in ATG7 KO mice. TB increased mtDNA content in the border zone significantly. In NRVMs subjected to I/R, TB reduced cell death by 41±6% (n=12, p&lt;0.001), decreased ROS, and increased mtDNA content significantly by ~50%. Moreover, TB promoted expression of PGC1α (a major driver of mitochondrial biogenesis) both in the infarct border zone and NRVMs subjected to I/R by ~40%, and increased levels of mitochondrial dynamics gene transcripts Drp1, Fis1, and MFN1 / 2. Conversely, ATG7 knockdown in NRVMs and cardiac ATG7 KO abolished the beneficial effects of TB on mitochondria DNA content. Conclusions: Autophagic flux is an essential process to mitigate myocardial reperfusion injury acting, at least in part, by inducing PGC1α-mediated mitochondrial biogenesis. Augmentation of autophagic flux may emerge as a viable clinical therapy to reduce reperfusion injury in myocardial infarction.

Abstract 13614: Nck1 Regulates Endothelial Activation and Blood Brain Integrity After Ischemia Reperfusion Injury

Introduction: Stroke remains a leading cause of disease disability worldwide. Rapid restoration of blood flow after occlusion is required to minimize brain damage. However, with the reperfusion there is upregulation to oxidative stress pathways that stimulates interleukin-1 activates kinase type I (IRAK-1) within endothelial cells leading to proinflammatory genes expression (ICAM-1/VCAM-1). We previously showed that the Nck family of adaptor proteins promotes oxidative stress-induced inflammation following ischemia reperfusion (IRI). Nck1 and Nck2 are two highly similar proteins but have inherent functional differences. Hypothesis: We hypothesized that Nck1 is required for IRAK-1 activation in response to IRI. Methods: Human brain endothelial cells (ECs) were genetically silenced for Nck1, Nck2, or interleukin-1 receptor type I kinase-1 (IRAK-1) and exposed to hydrogen peroxide (exogenous oxidative stress) or hypoxia re-oxygenation (endogenous oxidative stress). Additionally, adult male mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) followed by 24h reperfusion. Neurological deficits were assessed using the 24 sensorimotor scoring system. Infarct volume, blood brain barrier (BBB) integrity, and brain inflammatory profiles were assessed using immunostaining and immunoblotting. Results: Knock down of endothelial Nck1 or Nck2 of cells markedly reduced VCAM-1 and ICAM-1 expression and NF-κB activation in response to H 2 O 2 and hypoxia re-oxygenation. In ECs treated with the proinflammatory mediator interleukin-1 (IL-1β), there was upregulation of IRAK-1 and NF-κB activation. However, deletion of Nck1, but not Nck2, significantly decreased IRAK-1/ NF-κB activation by IL-1β. Moreover, Nck1 inhibition decreased endothelial permeability-induced by oxidative stress. In response to oxidative stress, Nck1 directly binds to IRAK-1. Nck1 KO mice had significantly less infarct volume and neurological deficits after MCAO/reperfusion. BBB disruption measured by Evans Blue extravasation and Fibrinogen staining was remarkably reduced in Nck1 KO mice. Conclusions: Nck1 promotes ischemia/reperfusion-induced inflammation, and brain injury, therefore, Nck1 inhibition is a promising therapy to improve IRI.

Celecoxib-Dependent Neuroprotection in a Rat Model of Transient Middle Cerebral Artery Occlusion (tMCAO) Involves Modifications in Unfolded Protein Response (UPR) and Proteasome.

Stroke is one of the main causes of death and disability worldwide. Ischemic stroke results in unfolded/misfolded protein accumulation in endoplasmic reticulum (ER), a condition known as ER stress. We hypothesized that previously reported neuroprotection of celecoxib, a selective inhibitor of cyclooxygenase-2, in transient middle cerebral artery occlusion (tMCAO) model, relies on the ER stress decrease. To probe this hypothesis, Sprague-Dawley rats were subjected to 1h of tMCAO and treated with celecoxib or vehicle 1 and 24h after ischemia. Protein and mRNA levels of the main hallmarks of ER stress, unfolded protein response (UPR) activation, UPR-induced cell death, and ubiquitin proteasome system (UPS) and autophagy, the main protein degradation pathways, were measured at 12 and 48h of reperfusion. Celecoxib treatment decreased polyubiquitinated protein load and ER stress marker expression such as glucose-related protein 78 (GRP78), C/EBP (CCAAT/enhancer-binding protein) homologous protein (CHOP), and caspase 12 after 48h of reperfusion. Regarding the UPR activation, celecoxib promoted inositol-requiring enzyme 1 (IRE1) pathway instead of double-stranded RNA-activated protein kinase-like ER kinase (PERK) pathway. Furthermore, celecoxib treatment increased proteasome catalytic subunits transcript levels and decreased p62 protein levels, while the microtubule-associated protein 1 light chain 3 (LC3B) II/I ratio remained unchanged. Thus, the ability of celecoxib treatment on reducing the ER stress correlates with the enhancement of IRE1-UPR pathway and UPS degradation. These data support the ability of anti-inflammatory therapy in modulating ER stress and reveal the IRE1 pathway as a promising therapeutic target in stroke therapy.Graphical abstract.

The targets of β-sitosterol as a novel therapeutic against cardio-renal complications in acute renal ischemia/reperfusion damage.

This research is the first to use β-sitosterol on myocardial and renal tissues in renal ischemia/reperfusion (IR) damage. Female Wistar rats were randomly divided into three groups: control (sham), renal IR (50min ischemia - 3h reperfusion), and renal IR + 150mg/kg/p.o. β-sitosterol (the rats were treated with β-sitosterol orally once 1h before the IR procedure). β-Sitosterol pretreatment caused an increase in superoxide dismutase and glutathione activities and a decrease in malondialdehyde levels in the kidney and heart. Moreover, it alleviated histopathological changes and downregulated the levels of tumor necrosis factor-alpha and interleukin-6 and upregulated the levels of endothelial nitric oxide synthase. As conclusion, the potential of β-sitosterol for renal and cardiac necrosis and apoptosis appears to act by limiting inflammatory response and oxidative stress. Thus, the potential of this compound is noteworthy and may serve as a potential therapeutic in the treatment of acute organ damages due to renal IR.

The novel GLP-1/GIP dual agonist DA3-CH is more effective than liraglutide in reducing endoplasmic reticulum stress in diabetic rats with cerebral ischemia-reperfusion injury

Background and aimsDiabetes is one of the most important risk factors and comorbidities of ischemic stroke. Endoplasmic reticulum stress (ERS) is considered to be the major injury mechanism of ischemic stroke with diabetes. Studies have found that incretin can inhibit ERS in ischemia-reperfusion injury of the liver and heart. We aimed to explore the effects of GLP-1/GIP double agonist DA3-CH and GLP-1 single agonist liraglutide on ERS and apoptosis in diabetic rats with cerebral ischemia-reperfusion injury. Methods and results72 Sprague–Dawley (SD) male rats were randomly divided into 4 groups: ① blank group (Sham group, n = 18); model group (Saline group, n = 18); DA3 treatment group (DA3 group, n = 18); liraglutide treatment group (Lir group, n = 18). The Sham group was not given any treatment and was only raised in the same environment as the other groups. The remaining 3 groups used STZ-induced diabetes models. After the successful membrane formation of diabetes, DA3-CH and liraglutide (10 mmol/kg, once-daily for 14 days) were injected intraperitoneally. Thereafter, rats were subjected to middle cerebral artery occlusion followed by 24-h reperfusion. Animals were evaluated for neurologic deficit score, infarct volume, and biomarker analyses of the brain after ischemia. The DA3-CH-treated and liraglutide-treated groups showed significantly reduced scores of neurological dysfunction and cerebral infarction size, and reduced the expression of ERS markers GRP78, CHOP and Caspase-12, and the expression of apoptosis marker bax. Anti-apoptotic markers bcl-2 and neuronal numbers increased significantly. ConclusionsDA3-CH and liraglutide have obvious neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury with diabetes, which can reduce the infarct size and the neurological deficit score. Their exert neuroprotective effects in a rat model of cerebral ischemia-reperfusion injury with diabetes by inhibiting endoplasmic reticulum stress and thereby reducing apoptosis. DA3 is better than liraglutide.

Oxytocin Reduces Brain Injury and Maintains Blood-Brain Barrier Integrity After Ischemic Stroke in Mice.

The present study was designed to determine the effect of different doses of oxytocin (OXT) on neuronal injury, spatial memory, blood-brain barrier (BBB) integrity and to explore possible underlying molecular mechanisms in the early stage of stroke in mice. Stroke model was generated by middle cerebral artery occlusion (MCAO) for 60min and 24h reperfusion in mice. OXT at doses of 1, 2, 4 and 8IU/per mouse was administrated intranasally at the beginning of brain ischemia. Brain injury, BBB integrity, and spatial memory were evaluated by standard methods. Changes in the expressionof nuclear factor-kappa B (NF-κB), and TUNEL positive cell were detected by immunohistochemistry. The levels of vascular endothelial growth factor(VEGF), aquaporin-4 (AQP4) and brain-derived neurotrophic factor (BDNF) proteins were determined by western blotting and ELISA methods. OXT at doses of 4 and 8IU/per mouse reduced the infarct size by 42% and 52%, respectively, and improved spatial memory function (p < 0.001). OXT (8IU/per mouse) significantly reduced brain edema, BBB disruption and upregulated the AQP4 expression (p < 0.001). Finally, OXT significantly diminished the number of apoptotic, NF-κB positive cells and enhanced the expression of BDNF and VEGF proteins in the brain tissue (p < 0.001). These findings provide important evidences that OXT significantly suppresses neuronal damage in the early stage of stroke by inhibiting apoptotic and NF-κB signaling pathway, increasing the expression of VEGF, AQP4 and BDNF proteins and reducing the BBB leakage.

In Vivo Bioluminescence Imaging for Targeting Acute Hypoxic/Ischemic Small Intestine with Engineered Salmonella typhimurium

This study aimed at investigating the feasibility of bioluminescence imaging (BLI) with engineered Salmonella typhimurium (ΔppGpp S. typhimurium) for visualizing acute hypoxic/ischemic bowels. At the start of 12- or 24-h reperfusion, ΔppGpp S. typhimurium was injected into the lateral tail veins of rats in which three segments of the small intestine were respectively subjected to 2, 3, and 4 h of ischemia. BLI and magnetic resonance imaging were performed at each reperfusion time point. Bioluminescence was exclusively detected in the hypoxic/ischemic segment of the intestine, showing the ability of ΔppGpp S. typhimurium to specifically target and proliferate in a hypoxic/ischemic area. Serial monitoring of these rat models revealed a progressive increase in bacterial bioluminescence in the ischemic intestines in conjunction with viable bacterial counts. The viable bacterial counts were positively correlated with lactate dehydrogenase levels after 24 h of reperfusion following 3 or 4 h of ischemia as well as interleukin-6 levels after 24 h of reperfusion following 4 h of ischemia. Our findings demonstrated that BLI was able to detect the acute hypoxic/ischemic bowel via monitoring of the distribution, internalization, and activity of administered ΔppGpp S. typhimurium. These findings may be useful for the early diagnosis of ischemic bowel disease.

Protective Mechanisms of Suxiao Jiuxin Pills () on Myocardial Ischemia-Reperfusion Injury in vivo and in vitro.

ObjectiveTo study the protective mechanism of Chinese medicine Suxiao Jiuxin Pills (速效救心丸, SXJ) on myocardial ischemia and reperfusion (I/R) injury.MethodsMouse myocardial I/R injury model was created by 30-min coronary artery occlusion followed by 24-h reperfusion, the mice were then divided into the sham group (n=7), the I/R group (n=13), the tirofiban group (TIR, positive drug treatment, n=9), and the SXJ group (n=11). Infarct size (IS), risk region (RR), and left ventricle (LV) were analyzed with double staining methods. In addition, H9C2 rat cardiomyocytes were cultured with Na2S2O4 to simulate I/R in vitro. The phosphorylation of extracellular regulated protein kinases1/2 (ERK1/2), protein kinase B (AKT), glycogen synthase kinase-3β (GSK3β), and protein expression of GATA4 in nucleus were detected with Western blot assay.ResultsThe ratio of IS/RR in SXJ and TIR groups were lower than that in I/R group (SXJ, 22.4% ±6.6%; TIR, 20.8%±3.3%; vs. I/R, 35.4%±3.7%, P<0.05, respectively). In vitro experiments showed that SXJ increased the Na2S2O4-enhanced phosphorylation of AKT/GSK3β and nuclear expression of GATA4.ConclusionSXJ prevents myocardial I/R injury in mice by activating AKT/GSK3β and GATA4 signaling pathways.Electronic Supplementary MaterialSupplementary materials (Appendixes) are available in the online version of this article at 10.1007/s11655-020-2726-2.

Lung injury in brain ischemia/reperfusion is exacerbated by mechanical ventilation with moderate tidal volume in rats.

Ischemic stroke is one of the most frequent causes of injury in the central nervous system which may lead to multiorgan dysfunction, including in the lung. The aim of this study was to investigate whether brain ischemia/reperfusion with or without mechanical ventilation leads to lung injury. Male Sprague-Dawley rats were assigned to four groups: Sham, 1-h brain ischemia (MCAO)/24-h reperfusion (I/R), mechanical ventilation with moderate tidal volume (MTV), and I/R+MTV. The pulmonary capillary permeability (Kfc) was measured in the isolated perfused lung. Mean arterial blood pressure (MAP), heart rate (HR), blood-gas variables, histopathological parameters, lung glutathione peroxidase, and TNF-α were measured. Kfc in the I/R, MTV, and I/R+MTV groups were higher than that in the Sham group. In the I/R, MTV, and I/R+MTV groups, arterial partial pressures of oxygen and the arterial partial pressure of oxygen/fraction of inspired oxygen ratios were lower, whereas arterial partial pressures of carbon dioxide were higher than those in the Sham group. The histopathological score in the I/R group was more than that in the Sham group, and in the MTV and I/R+MTV groups were higher than those in the Sham and I/R groups. Furthermore, there were stepwise rises in TNF-α in the I/R, MTV, and I/R+MTV groups, respectively. There was no significant difference in MAP between groups. However, HR in the MTV group was higher than that in the Sham group. Brain ischemia/reperfusion leads to pulmonary capillary endothelial damage and the impairment of gas exchange in the alveolar-capillary barrier, which is exacerbated by mechanical ventilation with moderate tidal volume partially linked to inflammatory reactions.

PAQR3 protects against oxygen-glucose deprivation/reperfusion-induced injury through the ERK signaling pathway in N2A cells.

Cerebral ischemia-reperfusion injury is pivotal in the development of multiple-subcellular organelle and tissue injury after acute ischemic stroke. Recently, the Golgi apparatus (GA) has been shown to be a key subcellular organelle that plays an important role in neuroprotection against oxygen-glucose deprivation/reperfusion (OGD/R) injury. PAQR3, a scaffold protein exclusively localized in the GA, was originally discovered as a potential tumor suppressor protein. PAQR3 acts as a spatial regulator of Raf-1 that binds Raf-1 and sequesters it to the GA, where it negatively modulates the Ras/Raf/MEK/ERK signaling pathway in tumor models. Studies suggest that suppression of the ERK pathway can alleviate OGD/R-induced cell apoptosis. However, whether PAQR3 has potential effects on ischemic stroke and the underlying mechanism(s) remain unexplored. The current study is the first to show that PAQR3 was significantly downregulated in mouse neuroblastoma (N2A) cells upon OGD/R exposure, both at the mRNA and protein levels. Compared to that in controls, the mRNA level of PAQR3 began to decline at 0h (0h) after reperfusion, while the protein level began to decline at 4h. Furthermore, overexpression of PAQR3 reduced OGD/R-induced apoptosis. The mRNA and protein levels of total ERK1 and ERK2 were unaltered, while activated p-ERK1 and p-ERK2 were decreased in N2A cells transfected with a PAQR3 expression vector after OGD for 4h plus 24h of reperfusion. Collectively, these data indicated that increased PAQR3 expression protected against OGD/R-induced apoptosis possibly by inhibiting the ERK signaling pathway. Therefore, PAQR3 might be a new attractive target in the treatment of OGD/R insult, and the underlying mechanism will pave the way for its potential experimental and clinical application.

The Involvement of Mitochondrial Biogenesis in Selenium Reduced Hyperglycemia-Aggravated Cerebral Ischemia Injury.

Selenium has been shown to possess antioxidant and neuroprotective effects by modulating mitochondrial function and activating mitochondrial biogenesis. Our previous study has also suggested that selenium protected neurons against glutamate toxicity and hyperglycemia-induced damage by regulating mitochondrial fission and fusion. However, it is still not known whether the mitochondrial biogenesis is involved in selenium alleviating hyperglycemia-aggravated cerebral ischemia reperfusion (I/R) injury. The object of this study is to define whether selenium protects neurons against hyperglycemia-aggravated cerebral I/R injury by promoting mitochondrial biogenesis. In vitro oxygen deprivation plus high glucose model decreased cell viability, enhanced reactive oxygen species production, and meanwhile stimulated mitochondrial biogenesis signaling. Pretreated with selenium significantly decreased cell death and further activated the mitochondrial biogenesis signaling. In vivo 30min of middle cerebral artery occlusion in the rats under hyperglycemic condition enhanced neurological deficits, enlarged infarct volume, exacerbated neuronal damage and oxidative stress compared with normoglycemic ischemic rats after 24h reperfusion. Consistent to the in vitro results, selenium treatment alleviated ischemic damage in hyperglycemic ischemic animals. Furthermore, selenium reduced the structural changes of mitochondria caused by hyperglycemic ischemia and further promoted the mitochondrial biogenesis signaling. Selenium activates mitochondrial biogenesis signaling, protects mitochondrial structure integrity and ameliorates cerebral I/R injury in hyperglycemic rats.

Baicalein exerts neuroprotective effect against ischaemic/reperfusion injury via alteration of NF-kB and LOX and AMPK/Nrf2 pathway.

ischaemic stroke accounts for almost 11% of all deaths worldwide and has a high incidence of permanent disability among patients. Baicalein has many beneficial pharmacological properties, including anti-inflammatory and anti-oxidant effects. However, the neuroprotective effect of baicalein is still unclear. The current study scrutinizes the neuroprotective effect of baicalein against the ischaemic/reperfusion (I/R) injury via alteration of the nuclear factor kappa B (NF-kB) and AMP-activated protein kinase/nuclear factor erythroid 2-related factor 2 AMPK/Nrf2 signaling pathway. Wistar rats were used for the current study. In rats, I/R injury was caused by transient occlusion of the middle cerebral artery for 1h accompanied by reperfusion for 24h. The rats were divided into different groups and treated with the different doses of baicalein (2.5, 5 and 10mg/kg). The effects of baicalein on the murine neurological function were determined via infarct volume, neurological defect scores, and brain water content. The -inflammatory cytokines and oxidative stress were estimated in the region of the cortical along with the expression of apoptosis markers, such as B-cell lymphoma 2, Bax, and caspase-3. Quantitative reverse transcription polymerase chain reaction was used for the estimation of the NF-kB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression. Baicalein significantly (p < 0.001) ameliorated the infarction volume, brain water content, and neurological outcome, and the malondialdehyde level, and reduced the level of interleukin-1β, interleukin-6, tumor necrosis factor-α, superoxide dismutase, glutathione, and catalase in a dose-dependent manner. Baicalein significantly (p < 0.001) altered the expression of COX-2, PGE2, LOX-1 and NF-kB as compared to I/R control group rats. Baicalein significantly reduced the Nrf2 and AMPK levels, and protected the rat brain against the I/R injury, suggesting a neuroprotective effect via down-regulation of NF-kB and LOX-1 expression and the AMPK/Nrf2 pathway.

Antioxidant effect of p-coumaric acid on interleukin 1-β and tumor necrosis factor-α in rats with renal ischemic reperfusion

Background and aimsRenal ischemia-reperfusion occurs in some clinical conditions such as kidney surgery that can leads to acute renal failure. The aim of this study was to investigate the effect of p-coumaric acid (CA) on ischemia reperfusion (I/R) injury. MethodsThirty rats were randomly divided into five groups; control, CA (100mg/kg), I/R, propylene glycol (10%)+I/R and CA+I/R, (n=6 each). CA and propylene glycol were administered orally for 2 weeks. Then, the rats were subjected to bilateral renal ischemia for 45min and followed by reperfusion for 24h. All rats were killed and kidney function tests, tissue malondialdehyde and activity of antioxidant enzymes were determined. Histopathological evaluations were also performed. In addition, renal expression of the tumor necrosis factor-α and interleukin-1β were determined using enzyme-linked immunosorbent assay and immunohistochemistry. ResultsCA significantly improved the Cr and BUN levels in CA+I/R group compared to I/R group (p<0.005 and p<0.001, respectively). Reduction of tissue superoxide dismutase, glutathione peroxidase and catalase, were significantly improved by CA (p<0.01, p<0.01 and p<0.05). Treatment with CA also resulted in significant reduction in tissue MDA (p<0.05), TNF-α (p<0.001) and interleukin-1β expression (p<0.001) that were increased by renal I/R. Also, the rats treated with CA had nearly normal structure of the kidney. ConclusionsThe present findings suggest that, CA protects the kidneys against I/R injury via its antioxidant and anti-inflammatory effects.

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Role of MnSOD in AIFm2‐Mediated Apoptosis During Cardiac Ischemia/Reperfusion Injury

Simulated Stroke in a Mouse Neuronal Cell Culture Model

INTRODUCTIONDespite many efforts, stroke remains a cause of debilitating illness and death worldwide. The fact that not only ischemia itself, but also the return of blood flow aggravates cellular dysfunction, illustrates a clinical barrier. Hence, catheter‐based interventions, the therapy of ischemic strokes, can add detrimental tissue damage, described as ischemia/reperfusion (I/R) injury. It is therefore crucial to develop potential pharmacological strategies that attenuate reperfusion injury and improve neurological function. Animal in‐vivo and in‐vitro models are vital to determine potential targets for pharmacological therapies. While in‐vivo models better mimic the complexity of the clinical context, in‐vitro studies allow to focus on the pathophysiology and can be performed on individual cell types. This study aimed to establish an in‐vitro stroke model by investigating the effect of simulated I/R injury on mouse brain isolated neurons.METHODSNeurons were plated and grown to confluency. To simulate stroke, cells were then randomized to either control/normoxic (21% O2, 5% CO2, 74% N2) or hypoxic conditions in a hypoxic chamber (0.01% O2, 5% CO2, 94.99% N2) in serum‐ and glucose‐free media. Durations of hypoxia for 2.5, 5 and 10h were compared. After 2h of reoxygenation in regular media and normoxic culture conditions following hypoxia/normoxia, samples were assayed for cell number/viability, cytotoxicity by lactate dehydrogenase (LDH) release and mitochondrial viability. Statistics: Mann‐Whitney Rank Sum Test and regression analysis; alpha=0.05.RESULTSHypoxia significantly impaired cell number/viability after 5 and 10h, whereas 2.5h of hypoxia did not. A large decrease of cell number/viability was observed after 10h of hypoxia compared to 5h and 2.5h. The same observations were made for mitochondrial viability, apart from a slightly lower decrease after 5h hypoxia compared to the cell number/viability. Inverse relations were found for LDH release, except for an already significant increase in cytotoxicity after 2.5h of hypoxia.CONCLUSIONSOur data demonstrate that hypoxic injury in this model is time‐dependent. While 2.5h did not result in sufficient damage and 10h injured neurons too excessively, 5h hypoxia led to a moderate enough damage, still allowing for potential attenuation through protective strategies. Therefore, 5h hypoxia, followed by 2h reperfusion, appears to be a suitable duration for an in‐vitro mouse neuron stroke model to test new treatments.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&amp;D Service.

Comparison of Continuous Hypothermic Oxygenated Crystalloid Perfusion with the Novel Solution “Custodiol N ” and Warm Blood Perfusion in a Porcine DCD Donation Model

Transplantation programs are worldwide burdened by the limited availability of suitable donor hearts, despite extending the donation criteria. Organ donation after circulatory death (DCD) became a reliable alternative over the past few years. Nevertheless, the grafts fetched as DCDs are submitted to a further ischemic insult due to the progressive hypoxia and circulatory arrest occurring between withdrawal of care and declaration of death. Although diverse storage and perfusion strategies exist to preserve DCD hearts, no direct comparison has yet been performed. The aim of the present study is to examine the effects of a novel cold crystalloid preservation solution (Custodiol-N) and warm blood perfusion protocols compared to simple cold storage in a porcine model. In 39 pigs, DCD was induced by asphyxia and the hearts underwent 30 min initial warm ischemia in the donor. The hearts were than explanted and divided in 4 groups: 1) control group (n=8) was placed directly in a Langendorff model. In group 2 (CSG, n=8) the hearts were flushed with 2L of 4°C Custodiol N solution and then stored in the same solution for 4h before evaluation. In group 3 (CCP, n=8), the hearts underwent a 4h continuous oxygenated 4°C cold machine perfusion cycle with Custodiol N. In group 4 (WBP, n=7) the hearts were placed on warm blood reperfusion for 4h. Left ventricular function was defined by dP/dt min & max at different ventricle filling volumes. Coronary blood flow (CBF) was measured at 100 mmHg perfusion pressure and Troponin T was collected up to 2 hours. Both dP/dt min & max were significantly higher in the CPP group compared to the WBP group [-1038±182 vs -344±38 mmHg/s; p=0.0356 and 1441±194 vs 498±53 mmHg/s; p=0.0123 respectively]. CBF was also significantly higher in the CCP group [1145±57vs 726±72ml/min; p=0.0032]. Moreover, Troponin T was significantly elevated in the WBP compared to the CCP group [27477±11777 vs 251034±90247 pg/ml, p=0.0239]. There were no significant differences among the WBP, CSG and control groups. Continuous hypothermic oxygenated crystalloid perfusion with the novel preservation solution Custodiol N offers superior preservation and resuscitation of DCD hearts in terms of functional recovery and myocardial enzyme release.

Beneficial consequences of Lupeol on middle cerebral artery-induced cerebral ischemia in the rat involves Nrf2 and P38 MAPK modulation.

Lupeol has been reported to exhibit anti-inflammatory and anti-tumor activities in many diseases, but its potential effects in cerebral ischemia injury have not been studied to date. In this work we present evidence for a beneficial effect of lupeol in a rat model of middle cerebral artery occlusion (MCAO) followed by reperfusion (MCAO/R) injury and provide some histological and biochemical evidence for its mechanism of action. A cerebral MCAO rat model was established by vascular occlusion for 2h, followed by 24h reperfusion period. The infarct volume, neurological deficits, and brain water content were compared with animals treated during reperfusion with different concentrations of lupeol. Macroscopic parameters, cell viability, pro-inflammatory factors generation, as well as oxidative stress parameters and associated apoptotic signaling cascades were evaluated. Treatment with lupeol significantly reduced the cerebral infarct volume and water content and recovered neuro behavioral functions in affected rats. Lupeol treatment down-regulated the expression of oxidative stress and inflammation factors. In addition, lupeol activated Nrf2, suppressed caspase-3 activity, reduced BAX/Bcl-2 ratio and inhibited phosphorylation of p38 MAPK. The data suggest that lupeol may exert protective effects against cerebral ischemia by suppressing oxidative stress and reduction of inflammation factors possible via activation of nuclear transcription factors and inhibition of cell death pathways.