Ischemia-reperfusion Insult Research Articles

Targeting S100A9-TLR2 axis controls macrophage NLRP3 inflammasome activation in fatty liver ischemia reperfusion injury

Abstract Liver ischemia reperfusion (IR) injury significantly impacts clinical outcomes by increasing the risk of hepatic dysfunction after liver surgery. Fatty livers are more susceptible to IR stress. Recent studies have demonstrated that S100A9 plays a crucial role in both IR injury and the progression of liver steatosis. Nevertheless, the precise mechanisms underlying these effects remain unclear. In our study, transcriptome analysis of fatty livers subjected to IR insult in mice identified S100A9 as an important mediator. Employing loss-of-function approaches, we investigated the immune regulatory function of S100A9 and its downstream signaling in fatty liver IR injury. As expected, S100A9 emerged as one of the most significantly upregulated genes during the reperfusion stage in fatty livers. Genetic knockdown of S100A9 markedly ameliorated liver pathological damage, evidenced by reduced macrophage/neutrophil infiltration as well as the decreased expression of proinflammatory factors. Transcriptome/functional studies revealed that S100A9 triggered liver inflammatory response via regulating Toll-like receptor 2 (TLR2)/Activating transcription factor 4 (ATF4) signaling. Additionally, TLR2 expression was notably increased in macrophages from ischemic fatty livers. In vitro, recombinant S100A9-stimulated macrophages exhibited the elevated production of proinflammatory factors and TLR2/ATF4 pathway activation. Intriguingly, S100A9 facilitated ATF4 nuclear translocation and enhanced NEK7/NLRP3 inflammasome activation in macrophages. In conclusion, our study identified S100A9 as a key regulator responsible for macrophage NLRP3 inflammasome activation and subsequent inflammatory injury in fatty liver IR process. Targeting TLR2/ATF4 signaling may offer a novel therapeutic strategy for mitigating S100A9-mediated liver injury.

S100A9 regulates M1 macrophage polarization and exacerbates steatotic liver ischemia-reperfusion injury

ObjectiveSteatotic livers exhibit higher susceptibility to ischemia reperfusion (IR) injury, which increase the risk of primary graft non-function following liver transplantation. S100A9 is identified as a pivotal innate immune sensor that regulates the progression of liver diseases. However, its significance in steatotic liver IR injury remains under-investigated. MethodsIn mice model, we generated S100A9 knockout (S100A9 KO) mice to investigate the role of S100A9 in IR-stimulated steatotic livers. In vitro, primary bone marrow-derived macrophages were utilized to explore the effect of S100A9 in regulating macrophage polarization and inflammation. ResultsS100A9 expression was markedly increased in steatotic livers of mice subjected to IR insult. S100A9 deletion significantly attenuated liver inflammatory injury, as evidenced by the diminished infiltration of both monocytes/macrophages and neutrophils (p < 0.05). The expression of proinflammatory factors was reduced (p < 0.05) at the same time. Additionally, S100A9-deficient livers demonstrated M1 polarization decrease and Toll-like receptor 4 (TLR4) suppression (p < 0.05). In vitro, genetic TLR4 inhibition led to nuclear factor kappa B (NF-κB) inactivation and subsequent M1 polarization decrease (p < 0.05) in macrophages treated with recombinant S100A9. ConclusionIn this study, we highlight the pivotal role of TLR4/NF-κB as a critical mediator of S100A9 in inducing M1 macrophage polorization- dependent inflammation in steatotic livers IR injury.

Cerebral Ischemia-Reperfusion Induced Neuronal Damage, Inflammation, miR-374a-5p, MAPK6, NLRP3, and Smad6 Alterations: Rescue Effect of N-acetylcysteine

Background: Ischemic stroke (IS) is still a major cause of neurological disability. This study aimed to ascertain potential markers closely related to IS diagnosis and treatment and then we examined the neuroprotective effect of N-acetylcysteine (NAC) in a transient cerebral ischemia. Methods: Male Wistar rats were randomly divided into three groups (n=6), including sham, IR (ischemia-reperfusion), IR+NAC (150 mg/kg, ip; intraperitoneally, 1 hour prior to ischemia and 5 min before reperfusion). The infarct volume was evaluated by 2,3,5-triphenyl tetrazolium chloride staining. H&amp;E and Nissle staining were performed to evaluate cerebral ischemia-reperfusion injury. miR-374a-5p gene expression, MAPK6, NLRP3, smad6, TNF-α, and IL-1β protein levels were determined by Real-time PCR, Western blot, and Elisa in the cerebral cortex exposed to IR. Results: Herein, we found that IR increased infarct volume and pathological damage to the cerebral cortex after global cerebral artery occlusion/reperfusion. In addition, miR-374a-5p gene expression decreased, while MAPK6, NLRP3, and smad6 protein expressions increased in the IR group. TNF-α and IL-1β protein levels increased in the ischemic cortex. Treatment with NAC significantly attenuated infarct size, inflammation and reversed aforementioned molecule levels. Conclusion: Taken together, these results suggested that ischemic insult can increase infarct size, neuronal damage, and inflammation may in part by modulating miR-374a-5p, MAPK6, NLRP3, and smad6 pathway in the brain cortex after cerebral IR insult and providing new clues to molecular mechanisms and treatment targets in IS. It can be alleviated by NAC as a potential therapy for someone afflicted with ischemia.

Differential Mitochondrial Bioenergetics in Neurons and Astrocytes Following Ischemia-Reperfusion Injury and Hypothermia.

The close interaction between neurons and astrocytes has been extensively studied. However, the specific behavior of these cells after ischemia-reperfusion injury and hypothermia remains poorly characterized. A growing body of evidence suggests that mitochondria function and putative transference between neurons and astrocytes may play a fundamental role in adaptive and homeostatic responses after systemic insults such as cardiac arrest, which highlights the importance of a better understanding of how neurons and astrocytes behave individually in these settings. Brain injury is one of the most important challenges in post-cardiac arrest syndrome, and therapeutic hypothermia remains the single, gold standard treatment for neuroprotection after cardiac arrest. In our study, we modeled ischemia-reperfusion injury by using in vitro enhanced oxygen-glucose deprivation and reperfusion (eOGD-R) and subsequent hypothermia (HPT) (31.5 °C) to cell lines of neurons (HT-22) and astrocytes (C8-D1A) with/without hypothermia. Using cell lysis (LDH; lactate dehydrogenase) as a measure of membrane integrity and cell viability, we found that neurons were more susceptible to eOGD-R when compared with astrocytes. However, they benefited significantly from HPT, while the HPT effect after eOGD-R on astrocytes was negligible. Similarly, eOGD-R caused a more significant reduction in adenosine triphosphate (ATP) in neurons than astrocytes, and the ATP-enhancing effects from HPT were more prominent in neurons than astrocytes. In both neurons and astrocytes, measurement of reactive oxygen species (ROS) revealed higher ROS output following eOGD-R, with a non-significant trend of differential reduction observed in neurons. HPT after eOGD-R effectively downregulated ROS in both cells; however, the effect was significantly more effective in neurons. Lipid peroxidation was higher after eOGD-R in neurons, while in astrocytes, the increase was not statistically significant. Interestingly, HPT had similar effects on the reduction in lipoperoxidation after eOGD-R with both types of cells. While glutathione (GSH) levels were downregulated after eOGD-R in both cells, HPT enhanced GSH in astrocytes, but worsened GSH in neurons. In conclusion, neuron and astrocyte cultures respond differently to eOGD-R and eOGD-R + HTP treatments. Neurons showed higher sensitivity to ischemia-reperfusion insults than astrocytes; however, they benefited more from HPT therapy. These data suggest that given the differential effects from HPT in neurons and astrocytes, future therapeutic developments could potentially enhance HPT outcomes by means of neuronal and astrocytic targeted therapies.

Protective Effects of Trans-Chalcone on Myocardial Ischemia and Reperfusion Challenge through Targeting Phosphoinositide 3-kinase/Akt-inflammosome Interaction.

Ischemia-reperfusion (IR) injury remains a pivotal contributor to myocardial damage following acute coronary events and revascularization procedures. Phosphoinositide 3-kinase (PI3K), a key mediator of cell survival signaling, plays a central role in regulating inflammatory responses and cell death mechanisms. Trans-chalcone (Tch), a natural compound known for its anti-inflammatory activities, has shown promise in various disease models. The aim of the current study was to investigate the potential protective effects of Tch against myocardial injury induced by ischemia and reperfusion challenges by targeting the PI3K-inflammasome interaction. Experimental models utilizing male rats subjected to an in vivo model of IR injury and myocardial infarction were employed. Administration of Tch (100 μg/kg, intraperitoneally) significantly reduced myocardial injury, as indicated by limited infarct size and decreased levels of the myocardial enzyme troponin. Mechanistically, Tch upregulated PI3K expression, thereby inhibiting the activity of the NOD-like receptor protein 3 inflammasome followed by the activation of pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. Moreover, it mitigated oxidative stress and suppressed vascular-intercellular adhesion molecules, contributing to its cardioprotective effects. The PI3K/Akt pathway inhibitor LY294002 considerably attenuated the beneficial effects of Tch. These findings highlight the therapeutic potential of Tch in ameliorating myocardial injury associated with IR insults through its modulation of the PI3K/Akt-inflammasome axis. The multifaceted mechanisms underlying its protective effects signify Tch as a promising candidate for further exploration in developing targeted therapies aimed at mitigating ischemic heart injury and improving clinical outcomes in cardiovascular diseases characterized by IR injury.

Parental obesity predisposes offspring to kidney dysfunction and increased susceptibility to ischemia-reperfusion injury in a sex-dependent manner.

Although obesity is recognized as a risk factor for cardiorenal and metabolic diseases, the impact of parental obesity on the susceptibility of their offspring to renal injury at adulthood is unknown. We examined the impact of parental obesity on offspring kidney function, morphology, and markers of kidney damage after acute kidney injury (AKI). Offspring from normal (N) diet-fed C57BL/6J parents were fed either N (NN) or a high-fat (H) diet (NH) from weaning until adulthood. Offspring from obese H diet-fed parents were fed N (HN) or H diet (HH) after weaning. All offspring groups were submitted to bilateral AKI by clamping the left and right renal pedicles for 30 min. Compared with male NH and NN offspring from lean parents, male HH and HN offspring from obese parents exhibited higher kidney injury markers such as urinary, renal osteopontin, plasma creatinine, urinary albumin excretion, and neutrophil gelatinase-associated lipocalin (NGAL) levels, and worse histological injury score at 22 wk of age. Only albumin excretion and NGAL were elevated in female HH offspring from obese parents compared with lean and obese offspring from lean parents. We also found an increased mortality rate and worse kidney injury scores after AKI in male offspring from obese parents, regardless of the diet consumed after weaning. Female offspring were protected from major kidney injury after AKI. These results indicate that parental obesity leads to increased kidney injury in their offspring after ischemia-reperfusion in a sex-dependent manner, even when their offspring remain lean.NEW & NOTEWORTHY Offspring from obese parents are more susceptible to kidney injury and worse outcomes following an acute ischemia-reperfusion insult. Male, but not female, offspring from obese parents exhibit increased blood pressure early in life. Female offspring are partially protected against major kidney injury induced by ischemia-reperfusion.

A small molecule macrophage migration inhibitory factor agonist ameliorates age-related myocardial intolerance to ischemia-reperfusion insults via metabolic regulation

Macrophage migration inhibitory factor (MIF) is an innate cytokine that regulates both inflammatory and homeostatic responses. MIF is expressed by cardiomyocytes, where it exerts a protective action against ischemia-reperfusion (I/R) injury by activating AMP-activated protein kinase (AMPK). This effect is attenuated in the senescent heart due to an intrinsic, age-related reduction in MIF expression. We hypothesized that treating the aged heart with the small molecule MIF agonist (MIF20) can reinforce protective MIF signaling in cardiomyocytes, leading to a beneficial effect against I/R stress. The administration of MIF20 at the onset of reperfusion was found to not only decrease myocardial infarct size but also preserves systolic function in the aged heart. Protection from I/R injury was reduced in mice with cardiomyocyte-specific Mif deletion, consistent with the mechanism of action of MIF20 to allosterically increase MIF affinity for its cognate receptor CD74. We further found MIF20 to contribute to the maintenance of mitochondrial fitness and to preserve the contractile properties of aged cardiomyocytes under hypoxia/reoxygenation. MIF20 augments protective metabolic responses by reducing the NADH/NAD ratio, leading to a decrease in the accumulation of reactive oxygen species (ROS) in the aged myocardium under I/R stress. We also identify alterations in the expression levels of the downstream effectors PDK4 and LCAD, which participate in the remodeling of the cardiac metabolic profile. Data from this study demonstrates that pharmacologic augmentation of MIF signaling provides beneficial homeostatic actions on senescent myocardium under I/R stress.

Hydroxycitric Acid Alleviated Lung Ischemia-Reperfusion Injury by Inhibiting Oxidative Stress and Ferroptosis through the Hif-1α Pathway.

Lung ischemia-reperfusion injury (LIRI) is a prevalent occurrence in various pulmonary diseases and surgical procedures, including lung resections and transplantation. LIRI can result in systemic hypoxemia and multi-organ failure. Hydroxycitric acid (HCA), the primary acid present in the peel of Garcinia cambogia, exhibits anti-inflammatory, antioxidant, and anticancer properties. However, the effects of HCA on LIRI remain unknown. To investigate the impact of HCA on LIRI in mice, the mice were randomly divided into four groups: the control group, the I/R model group, and the I/R + low- or high-dose HCA groups. Human umbilical vein endothelial cells (HUVECs) were subjected to hypoxia for 12 h followed by reoxygenation for 6 h to simulate in vitro LIRI. The results demonstrated that administration of HCA effectively attenuated lung injury, inflammation, and edema induced by ischemia reperfusion. Moreover, HCA treatment significantly reduced malondialdehyde (MDA) and reactive oxygen species (ROS) levels while decreasing iron content and increasing superoxide dismutase (SOD) levels after ischemia-reperfusion insult. Mechanistically, HCA administration significantly inhibited Hif-1α and HO-1 upregulation both in vivo and in vitro. We found that HCA could also alleviate endothelial barrier damage in H/R-induced HUVECs in a concentration-dependent manner. In addition, overexpression of Hif-1α counteracted HCA-mediated inhibition of H/R-induced endothelial cell ferroptosis. In summary, these results indicate that HCA alleviated LIRI by inhibiting oxidative stress and ferroptosis through the Hif-1α pathway.

Conditioned Medium Derived From the Human Amniotic Membrane Prevents Brain Damage Against Cerebral Ischemia/Reperfusion in Subacute, Acute, and Chronic Phases in a Rat Model of Stroke.

Stem cells isolated from the amniotic membrane can produce and release substances that can regenerate damaged tissues and contain proteins and other factors that via numerous major and minor mechanisms lead to increasing angiogenesis and tissue survival. This research was conducted to prove the defensive characteristics of the secretome in the face of temporary focal cerebral ischemia in mouse stroke models. Cerebral ischemia protocol in a specific area was implemented in rats with middle cerebral artery occlusion for 60 minutes and then reperfusion was given for 6, 20, and 30 minutes. Within 30 minutes after the start of reperfusion, conditioned medium derived from the human amniotic membrane (AMSC-CM) was poured into the right ventricle (ICV) at a dose of 0.5 μL. Finally, the volume of the injury, cerebral tissue water, sensorimotor activity, and the strength of the blood-brain barrier integrity were evaluated 24 hours after drug injection. ICV injection of conditioned medium at the start of reperfusion phase considerably decreased the volume of the injury 6, 20, and 30 hours after reperfusion compared to the MCAO-operated group (P<0.01). Cerebral tissue water in the treatment group decreased considerably after the intervention in comparison with the MCAO group in the core and penumbral area not in the subcortical area (P<0.05). Also, the amount of Evans blue infiltration at all times in the core and half-foot area in the AMSC-CM group was significantly reduced in parallel with the MCAO group (P<0.05). Treatment with AMSC-CM during 6-30 h after ischemia-reperfusion insult exerts some beneficial effects against ischemia-reperfusion injury. These findings provide an important vision for more complementary research and treatment of stroke. During ischemia, the brain undergoes a series of harmful pathological events.Mesenchymal stem cells culture medium is called condition medium (CM).CM contains substances that have neuroprotective properties.The use of CM reduces the damage caused by cerebral ischemia. The stem cell culture medium is called condition medium (CM). CM contains substances that have neuroprotective properties. This study aims to investigate the effect of CM on injuries caused by stroke. After causing a stroke in rats by closing the artery, the CM was injected into the injury site. The results showed that CM reduced the severity of the injury.

Cdk1 protects against oxygen-glucose deprivation and reperfusion-induced Golgi fragmentation and apoptosis through mediating GM130 phosphorylation.

Increasing evidence has indicated that the Golgi apparatus (GA) is involved in the development of cerebral ischemia-reperfusion (IR) injury. Finding effective neuroprotective agents targeting GA has become a priority in the treatment of ischemic stroke. GM130, a key structural protein present on the cis-face of the GA, maintains its structure through its phosphorylation and dephosphorylation. However, the molecular mechanisms by which GM130 regulates IR-induced neuronal apoptosis are not well elucidated. Mouse neuroblastoma Neuro2a (N2A) cells were subjected to oxygen-glucose deprivation and reperfusion (OGDR) insult. Cell proliferation and apoptosis were determined using MTT assay, TUNEL staining, and flow cytometry. GA morphology was detected by immunocytochemical staining and immunofluorescence microscopy. GA-related protein and mRNA levels were detected by WB and qPCR, respectively. Treatment with Purvalanol A, an effective Cdk1 inhibitor, and transfection of Cdk1-shRNA were carried out to inhibit OGDR-induced Cdk1 elevation. The results demonstrated that OGDR induced Golgi fragmentation, neuronal apoptosis, GM130 phosphorylation, and p115 cleavage in N2A cells. Cdk1 elevation after OGDR was closely correlated with GM130 phosphorylation, not p115. Inhibition of Cdk1 significantly attenuated OGDR-induced Golgi fragmentation and cell apoptosis. Cdk1 interacted with GM130 and decreased its phosphorylation on the serine 25 site in N2A cells exposed to OGDR. The present findings reveal that Cdk1 protects against IR-induced GA fragmentation and apoptosis, likely through the mediation of GM130 phosphorylation. This neuroprotective potential of Cdk1 against IR insult and the underlying mechanism will pave the way for potential clinical applications targeting the GA organelle for cerebral IR-related disorders.

Abstract P397: Preservation Of Renal Microvascular Function In Ischemia-reperfusion Induced Kidney Injury In Rats

Proper renal blood flow and glomerular filtration rate are critical for maintaining normal kidney function by precisely adjusting preglomerular microvascular resistance. Renal ischemia-reperfusion (IR) insult markedly increased mRNA expression of the inflammatory chemokine (C-C motif) Ligand 2 (CCL2) in rat kidney tissues. We hypothesized that depletion of macrophages prevented IR-induced renal autoregulatory impairment via reduction of CCL2 in preglomerular microvessels (PGMV). IR was induced in male rats by 60-minute bilateral renal artery occlusion followed by reperfusion. Some IR rats were treated with the selective macrophage inhibitor, Liposome-encapsulated clodronate (LC) following reperfusion and repeated 72 hours post-IR. Afferent arteriolar autoregulatory reactivity was assessed using the in vitro blood-perfused juxtamedullary nephron preparation in sham, IR, and IR+LC rats on day 7. Afferent arterioles from sham-operated rats exhibited pressure-dependent vasoresponsiveness. The baseline arteriolar diameter averaged 13.4 ± 0.5 μm at renal perfusion pressure (RPP) of 100 mmHg. Diameter increased to 117 ± 4% of baseline at RPP of 65 mmHg and decreased to 69 ± 2% at 170 mmHg, respectively. In contrast, afferent arterioles from IR rats only increased to 104 ± 2% and decreased to 91 ± 5% of baseline at 65 and 170 mmHg, respectively (P &lt; 0.05 vs sham), indicating the impaired autoregulatory capacity in IR rats. LC-treated IR rats, however, markedly improved the pressure-dependent vasoresponsiveness. A decrease in RPP to 65 mmHg resulted in a 124 ± 17% increase in the baseline diameter, whereas increasing RPP to 170 mmHg led to a 74 ± 7% decrease in diameter. Additionally, IR markedly increased CCL2 mRNA expression in isolated PGMV by 10-fold 7 days post-IR. IR also elevated NADPH oxidase subunits (gp91 phox , P67 phox , and P47 phox ). Significantly, LC treatment suppressed mRNA expression of CCL2, gp91 phox , P67 phox , and P47 phox in IR-PGMV. In conclusion, depletion of macrophages preserves afferent arteriolar autoregulatory capacity and reduces PGMV CCL2 mRNA expression in IR rats, suggesting a critical role of macrophages in mediating renal microvascular autoregulatory impairment in IR-induced kidney injury via the increase of CCL2 in PGMV.

Transition ofResuscitative Endovascular Balloon Occlusion ofthe Aorta from Zone 3 to Zone 1 to Treat Hemodynamic Collapse during Continued Hemorrhage.

Noncompressible torso hemorrhage (NCTH) accounts for most potentially survivable deaths on the battlefield. Treatment of NCTH is challenging, especially in far-forward environments with limited capabilities. Resuscitative endovascular balloon occlusion of the aorta (REBOA) has shown promise in the care of patients with NCTH. REBOA involves introducing a balloon catheter into the descending aorta in a specific occlusion region (zones 1, 2, or 3) and acts as a hemorrhage control adjunct with resuscitative support. The balloon is placed in zone 3 in the infrarenal aorta for high junctional or pelvic injuries and in zone 1 proximal to the diaphragm for torso hemorrhage. Zone 1 REBOA provides more resuscitative support than zone 3; however, the potential for ischemia and reperfusion injuries is greater with zone 1 than with zone 3 REBOA placement. This study aims to determine the possible benefit of transitioning the REBOA balloon from zone 3 to zone 1 to rescue a patient with ongoing venous bleeding and impending cardiovascular collapse. Yorkshire male swine (70-90 kg, n = 6 per group) underwent injury to the femoral artery, which was allowed to bleed freely for 60 s, along with a simultaneous controlled venous hemorrhage. After 60 s, the arterial bleed was controlled with hemostatic gauze and zone 3 REBOA was inflated. Five hundred milliliters of Hextend was used for initial fluid resuscitation. The controlled venous bleed continued until a mean arterial pressure (MAP) of 30 mmHg was reached to create an impending cardiovascular collapse. The animals were then randomized into either continued zone 3 REBOA or transition from zone 3 to zone 1 REBOA. Following 30 min, a "hospital phase" was initiated, consisting of cessation of the venous hemorrhage, deflation of the REBOA balloon, and transfusion of one unit of whole blood administered along with saline and norepinephrine to maintain a MAP of 60 mmHg or higher. The animals then underwent a 2-h observation period. Survival, hemodynamics, and blood chemistries were compared between groups. No significant differences between groups were observed in hemodynamic or laboratory values at baseline, postinitial injury, or when MAP reached 30 mmHg. Survival was significantly longer in animals that transitioned into zone 1 REBOA (log-rank analysis, P = .012). The average time of survival was 14 ± 10 min for zone 3 animals vs. 65 ± 59 min for zone 1 animals (P = .064). No animals in the zone 3 group survived to the hospital phase. Zone 1-treated animals showed immediate hemodynamic improvement after transition, with maximum blood pressure reaching near baseline values compared to those in the zone 3 group. In this swine model of NCTH, hemodynamics and survival were improved when the REBOA balloon was transitioned from zone 3 to zone 1 during an impending cardiovascular collapse. Furthermore, these improved outcome data support the pursuit of additional research into mitigating ischemia-reperfusion insult to the abdominal viscera while still providing excellent resuscitative support, such as intermittent or partial REBOA.

Furosemide exacerbated the impairment of renal function, oxygenation and medullary damage in a rat model of renal ischemia/reperfusion induced AKI

BackgroundPerioperative acute kidney injury (AKI) caused by ischemia–reperfusion (IR) is a significant contributor to mortality and morbidity after major surgery. Furosemide is commonly used in postoperative patients to promote diuresis and reduce tissue edema. However, the effects of furosemide on renal microcirculation, oxygenation and function are poorly understood during perioperative period following ischemic insult. Herein, we investigated the effects of furosemide in rats subjected IR insult.Methods24 Wistar albino rats were divided into 4 groups, with 6 in each; Sham-operated Control (C), Control + Furosemide (C + F), ischemia/reperfusion (IR), and IR + F. After induction of anesthesia (BL), supra-aortic occlusion was applied to IR and IR + F groups for 45 min followed by ongoing reperfusion for 15 min (T1) and 2 h (T2). Furosemide infusion was initiated simultaneously in the intervention groups after ischemia. Renal blood flow (RBF), vascular resistance (RVR), oxygen delivery (DO2ren) and consumption (VO2ren), sodium reabsorption (TNa+), oxygen utilization efficiency (VO2/TNa+), cortical (CμO2) and medullary (MμO2) microvascular oxygen pressures, urine output (UO) and creatinine clearance (Ccr) were measured. Biomarkers of inflammation, oxidative and nitrosative stress were measured and kidneys were harvested for histological analysis.ResultsIR significantly decreased RBF, mainly by increasing RVR, which was exacerbated in the IR + F group at T2 (2198 ± 879 vs 4233 ± 2636 dyne/s/cm5, p = 0.07). CμO2 (61.6 ± 6.8 vs 86 ± 6.6 mmHg) and MμO2 (51.1 ± 4.1 vs 68.7 ± 4.9 mmHg, p < 0.05) were both reduced after IR and did not improve by furosemide. Moreover, VO2/TNa+ increased in the IR + F group at T2 with respect to the IR group (IR: 3.3 ± 2 vs IR + F: 8.2 ± 10 p = 0.07) suggesting a possible deterioration of oxygen utilization. Ccr did not change, but plasma creatinine increased significantly in IR + F groups. Histopathology revealed widespread damage both in the cortex and medulla in IR, IR + F and C + F groups.ConclusionRenal microvascular oxygenation, renal function, renal vascular resistance, oxygen utilization and damage were not improved by furosemide administration after IR insult. Our study suggests that furosemide may cause additional structural and functional impairment to the kidney following ischemic injury and should be used with caution.

Targeting neuronal mitophagy in ischemic stroke: an update

Cerebral ischemia is a neurological disorder associated with complex pathological mechanisms, including autophagic degradation of neuronal mitochondria, or termed mitophagy, following ischemic events. Despite being well-documented, the cellular and molecular mechanisms underlying the regulation of neuronal mitophagy remain unknown. So far, the evidence suggests neuronal autophagy and mitophagy are separately regulated in ischemic neurons, the latter being more likely activated by reperfusional injury. Specifically, given the polarized morphology of neurons, mitophagy is regulated by different neuronal compartments, with axonal mitochondria being degraded by autophagy in the cell body following ischemia-reperfusion insult. A variety of molecules have been associated with neuronal adaptation to ischemia, including PTEN-induced kinase 1, Parkin, BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (Bnip3), Bnip3-like (Bnip3l) and FUN14 domain-containing 1. Moreover, it is still controversial whether mitophagy protects against or instead aggravates ischemic brain injury. Here, we review recent studies on this topic and provide an updated overview of the role and regulation of mitophagy during ischemic events.

Predictive factors for postrevascularization compartment syndrome in acute lower limb ischemia

Background Following revascularization treatments for acute limb ischemia, postischemic compartment syndrome (CS), a surgical emergency brought on by elevated intracompartmental pressure in a lower extremity, develops acute limb ischemia. The muscles in the extremities may become edematous during surgical revascularization, such as embolectomy or artery bypass, as a result of fluid extravasation or inflammatory reactions following an ischemia-reperfusion insult, leading to a fast rise in intracompartmental pressure. Objectives In patients with nontraumatic acute ischemia of the lower extremities, are to evaluate the risk variables for postrevascularization CS. Patients and methods The 50 patients for this study were chose n at random from the emergency hospital at Mansoura University’s Department of Vascular Surgery. Results The most important predictors of the CS were paralysis, Rutherford classification IIB, and intraoperative insufficient backflow. Significant risk factors for CS were atrial fibrillation, emboli, thrombosis, Rutherford classification IIA, ischemia time (h), and positive fluid balance in the first 24 h. Conclusion The most important predictors of the CS in postrevascularization of acute lower limb ischemia, as shown by the regression analysis, is paralysis, Rutherford classification IIB, and intraoperative insufficient backflow.

LncRNA nuclear-enriched abundant transcript 1 aggravates cerebral ischemia/reperfusion injury through activating early growth response-1/RNA binding motif protein 25 axis.

Ischemic stroke is a major global health issue. Ischemia and subsequent reperfusion results in stroke-related brain injury. Previous studies have demonstrated that nuclear-enriched abundant transcript 1 (NEATa and early growth response 1 (EGR1) are involved in ischemia reperfusion (IR) injury). In this study, we aimed to explore the roles of NEAT1/EGR1 axis as well as its downstream effector RNA binding motif protein 25 (RBM25) in cerebral IR injury. Oxygen-glucose deprivation/reperfusion (OGD/R) and middle cerebral artery occlusion (MCAO) were used to establish in vitro and in vivo models of cerebral IR injury, respectively. According to our data, NEAT1, EGR1, and RBM25 levels were elevated in OGD/R-exposed SK-N-SH and SH-SY5Y cells and cerebral cortex of MCAO mice. NEAT1, EGR1, or RBM25 knockdown effectively reduced infarct volumes and apoptosis, and improved neurological function. Mechanistically, NEAT1 directly interacted with EGR1, which restrained WW domain containing E3 ubiquitin protein ligase 1 (WWP1)-mediated ubiquitination of EGR1 and subsequently caused EGR1 accumulation. EGR1 bound to RBM25 promoter and transcriptionally activated RBM25. Rescue experiments indicated that RBM25 overexpression abolished the therapeutic effects of NEAT1 knockdown. In conclusion, this work identified a novel NEAT1/EGR1/RBM25 axis in potentiating brain injury after IR insults, suggesting a potential therapeutic target for ischemic stroke.

The LPA-CDK5-tau pathway mediates neuronal injury in an in vitro model of ischemia-reperfusion insult

Lysophosphatidic acid (LPA) is a common glycerol phospholipid and an important extracellular signaling molecule. LPA binds to its receptors and mediates a variety of biological effects, including the pathophysiological process underlying ischemic brain damage and traumatic brain injury. However, the molecular mechanisms mediating the pathological role of LPA are not clear. Here, we found that LPA activates cyclin-dependent kinase 5 (CDK5). CDK5 phosphorylates tau, which leads to neuronal cell death. Inhibition of LPA production or blocking its receptors reduced the abnormal activation of CDK5 and phosphorylation of tau, thus reversing the death of neurons. Our data indicate that the LPA-CDK5-Tau pathway plays an important role in the pathophysiological process after ischemic stroke. Inhibiting the LPA pathway may be a potential therapeutic target for treating ischemic brain injury.

Myristoylated Protein Kinase C Beta II Inhibitor Attenuates Renal Injury in Mice Subjected to Severe Bilateral Ischemia‐Reperfusion

Acute kidney injury (AKI) due to ischemia‐reperfusion (I/R) insult involves oxidative stress and inflammation leading to rapid renal decline and remains a significant cause of post‐operative mortality. Myristoylated protein kinase C beta II peptide inhibitor (N‐myr‐SLNPEWNET; myr‐PKCβII‐) is known to attenuate myocardial I/R injury in ex vivo rat hearts. We hypothesized that myr‐PKCβII‐ would attenuate severe renal I/R injury that is characterized by elevated serum creatinine (Cr) and a decrease in glomerular filtration rate (GFR). We predict that treatment with myr‐PKCβII‐ will improve these indices of kidney function compared to a scrambled control peptide (N‐myr‐WNPESLNTE; myr‐PKCβII‐scram).Renal pedicles of anesthetized male C57BL/6J mice (25–30g) were clamped bilaterally for 20 min or 19 min. Five minutes before unclamping, 2.0 mg/kg (20 µM serum) myr‐PKCβII‐ or myr‐PKCβII‐scram were given i.v. into the tail vein. Cr (mg/dL) was measured at baseline, 24h, 72h, and 96h post‐injury. GFR (µl/min) was determined with fluorescein‐isothiocyanate (FITC)‐Sinistrin renal clearance. Data were evaluated by unpaired Student’s t‐test.Following 20‐min renal ischemia (Fig 1.), myr‐PKCβII‐ (n=9) significantly reduced Cr at 24h and 72h post‐injury compared to myr‐PKCβII‐scram control (n=8, p&lt;0.05). However, there were three fatalities prior to 96h. In 19‐min ischemia, there were no fatalities up to 96h post‐injury. However, Cr levels of the myr‐PKCβII‐scram control (n=8) in 19‐min I/R were significantly lower than 20‐min I/R at all time points post‐injury (all p&lt;0.01): 24h (0.54 ± 0.10 vs 1.59 ± 0.06 mg/dL), 72h (0.24 ± 0.06 vs 1.28 ± 0.25 mg/dL), and 96h (0.17 ± 0.03 vs 0.72 ± 0.15 mg/dL). Myr‐PKCβII‐ did not significantly reduce Cr (Fig. 1) nor improve GFR (Fig. 2) following 19‐min I/R.Results suggest that 20‐min renal ischemia was more severe, which was indicated by a 5‐fold increase of peak Cr levels at 72h post‐injury compared to 19‐min ischemia and the unanticipated fatalities of three mice. Myr‐PKCβII‐ attenuated renal injury following 20‐min renal ischemia, but not at the milder 19‐min model in which peak Cr levels were too low to detect therapeutic benefit. The large difference in injury severity between 20‐min and 19‐min renal ischemia emphasizes the temporal relationship between renal function and ischemic duration.Future studies will characterize myr‐PKCβII‐ protection against AKI in 19‐min bilateral renal ischemia based on biomarkers indicating proximal renal tubule damage (e.g. NGAL and Kim‐1). A more optimal ischemic duration between 19 min and 20 min will be investigated for future experiments.

Sex-specific effects of prenatal hypoxia on the cardiac endothelin system in adult offspring.

Fetal hypoxia, a major consequence of complicated pregnancies, impairs offspring cardiac tolerance to ischemia-reperfusion (I/R) insult; however, the mechanisms remain unknown. Endothelin-1 (ET-1) signaling through the endothelin A receptors (ETA) is associated with cardiac dysfunction. We hypothesized that prenatal hypoxia exacerbates cardiac susceptibility to I/R via increased ET-1 and ETA levels, whereas ETA inhibition ameliorates this. Pregnant Sprague-Dawley rats were exposed to normoxia (21% O2) or hypoxia (11% O2) on gestational days 15-21. Offspring were aged to 4 mo, and hearts were aerobically perfused or subjected to ex vivo I/R, with or without preinfusion with an ETA antagonist (ABT-627). ET-1 levels were assessed with ELISA in aerobically perfused and post-I/R left ventricles (LV). ETA and ETB levels were assessed by Western blotting in nonperfused LV. As hypothesized, ABT-627 infusion tended to improve post-I/R recovery in hypoxic females (P = 0.0528); however, surprisingly, ABT-627 prevented post-I/R recovery only in the hypoxic males (P < 0.001). ET-1 levels were increased in post-I/R LV in both sexes regardless of the prenatal exposure (P < 0.01). ETA expression was similar among all groups, whereas ETB (isoform C) levels were decreased in prenatally hypoxic females (P < 0.05). In prenatally hypoxic males, ETA signaling may be essential for tolerance to I/R, whereas in prenatally hypoxic females, ETA may contribute to cardiac dysfunction. Our data illustrate that understanding the prenatal history has critical implications for treatment strategies in adult chronic diseases.NEW & NOTEWORTHY We demonstrated that prenatal hypoxia (a common condition of pregnancy) can have profound differential effects on treatment strategies in adult cardiovascular disease. Our data using a rat model of prenatal hypoxia demonstrated that, as adults, although inhibition of endothelin (ETA) receptors before an ex vivo cardiac ischemic insult improved recovery in females, it strikingly prevented recovery in males. Our data indicate a sex-specific effect of prenatal hypoxia on the cardiac ET-1 system in adult offspring.

Augmented Cardiac Mitochondrial Capacity in High Capacity Aerobic Running "Disease-Resistant" Phenotype at Rest Is Lost Following Ischemia Reperfusion.

Rationale: Regular active exercise is considered therapeutic for cardiovascular disease, in part by increasing mitochondrial respiratory capacity, but a significant amount of exercise capacity is determined genetically. Animal models, demonstrating either high capacity aerobic running (HCR) or low capacity aerobic running (LCR) phenotypes, have been developed to study the intrinsic contribution, with HCR rats subsequently characterized as “disease resistant” and the LCRs as “disease prone.” Enhanced cardioprotection in HCRs has been variable and mutifactoral, but likely includes a metabolic component. These studies were conducted to determine the influence of intrinsic aerobic phenotype on cardiac mitochondrial function before and after ischemia and reperfusion.Methods: A total of 34 HCR and LCR rats were obtained from the parent colony at the University of Toledo, housed under sedentary conditions, and fed normal chow. LCR and HCR animals were randomly assigned to either control or ischemia-reperfusion (IR). On each study day, one HCR/LCR pair was anesthetized, and hearts were rapidly excised. In IR animals, the hearts were immediately flushed with iced hyperkalemic, hyperosmotic, cardioplegia solution, and subjected to global hypothermic ischemic arrest (80 min). Following the arrest, the hearts underwent warm reperfusion (120 min) using a Langendorff perfusion system. Following reperfusion, the heart was weighed and the left ventricle (LV) was isolated. A midventricular ring was obtained to estimate infarction size [triphenyltetrazolium chloride (TTC)] and part of the remaining tissue (~150 mg) was transferred to a homogenation buffer on ice. Isolated mitochondria (MITO) samples were prepared and used to determine respiratory capacity under different metabolic conditions. In control animals, MITO were obtained and prepared similarly immediately following anesthesia and heart removal, but without IR.Results: In the control rats, both resting and maximally stimulated respiratory rates were higher (32 and 40%, respectively; p < 0.05) in HCR mitochondria compared to LCR. After IR, resting MITO respiratory rates were decreased to about 10% of control in both strains, and the augmented capacity in HCRs was absent. Maximally stimulated rates also were decreased more than 50% from control and were no longer different between phenotypes. Ca++ retention capacity and infarct size were not significantly different between HCR and LCR (49.2 ± 5.6 vs. 53.7 ± 4.9%), nor was average coronary flow during reperfusion or arrhythmogenesis. There was a significant loss of mitochondria following IR, which was coupled with decreased function in the remaining mitochondria in both strains.Conclusion: Cardiac mitochondrial capacity from HCR was significantly higher than LCR in the controls under each condition. After IR insult, the cardiac mitochondrial respiratory rates were similar between phenotypes, as was Ca++ retention capacity, infarct size, and arrhythmogenicity, despite the increased mitochondrial capacity in the HCRs before ischemia. Relatively, the loss of respiratory capacity was actually greater in HCR than LCR. These data could suggest limits in the extent to which the HCR phenotype might be “protective” against acute tissue stressors. The extent to which any of these deficits could be “rescued” by adding an active exercise component to the intrinsic phenotype is unknown.