Early Phase Of Reperfusion Research Articles

Controlled automated reperfusion of the whole body after cardiac arrest: Device profile of the CARL system.

Cardiac arrest is associated with high mortality rates and severe neurological impairments. One of the underlying mechanisms is global ischemia-reperfusion injury of the body, particularly the brain. Strategies to mitigate this may thus improve favorable neurological outcomes. The use of extracorporeal cardiopulmonary membrane oxygenation (ECMO) during CA has been shown to improve survival, but available systems are vastly unable to deliver goal-oriented resuscitation to control patient's individual physical and chemical needs during reperfusion. Recently, controlled automated reperfusion of the whoLe body (CARL), a pulsatile ECMO with arterial blood-gas analysis, has been introduced to deliver goal-directed reperfusion therapy during the post-arrest phase. This review focuses on the device profile and use of CARL. Specifically, we reviewed the published literature to summarize data regarding its technical features and potential benefits in ECPR. Peri-arrest, mitigating severe IRI with ECMO, might be the next step toward augmenting survival rates and neurological recovery. To this end, CARL is a promising extracorporeal oxygenation device that improves the early reperfusion phase after resuscitation.

#337 Renal 3D photoacoustic imaging and contrast-enhanced ultrasound to assess oxygenation and perfusion and to predict fibrosis after ischemia-reperfusion

Abstract Background and Aims Renal 3D photo-acoustic imaging (3D-PAI) and contrast-enhanced ultrasound (CEUS) are promising tools in mice models to assess repeatedly and non-invasively renal ischemia-reperfusion (RIR) consequences and damages, or their improvement by nephroprotective strategies like mild therapeutic hypothermia (mTH), at the early phase of reperfusion as well as in the chronic late phase. Method C57Bl6 mice underwent 15 minutes of unilateral renal vascular clamping, with body temperature at 37°C (RIR-37°C, n=7) or mTH at 34°C (RIR-34°C, n=7), or a sham procedure (Sham, n=5). Renal volume and oxygen saturation (sO2-3D; corresponding to the percentage of oxyhemoglobin over the total hemoglobin content) were measured with 3D-PAI (3D reconstruction of the whole kidney through multi-plane acquisition with an automatized mobile support), and renal perfusion parameters (rBV, mTT, rBF) with CEUS (“destruction-replenishment” model of intravenous microbubbles), performed with VEVO3100 echograph (Fujifilm Visualsonic) 1 week before RIR (basal), 20 minutes after reperfusion, and 1 month after. Renal fibrosis was quantified at 1 month with Masson's trichrome coloration on histological samples. Data were compared with Mann-Whitney test or Wilcoxon test (paired data) as appropriate. Correlation was tested by Spearman test. All animal procedures were approved by the Ethics Committee. Results Sham showed no significant changes during follow-up. RIR-37°C led to renal hypotrophy (volume on 3D-PAI) and fibrosis at 1 month, compared to RIR-34°C (median volume 76 IQR [71-119] vs 143 IQR[108-180] mm3; p=0.03), and those parameters were correlated (R=−0.49; p=0.03). In RIR-37°C, sO2-3D tended to lower at 20 minutes compared to basal values (median sO2-3D 37 IQR [37-44] vs 49 IQR [36-57]%; p=0.08), but not in RIR-34°C (p > 0.99), and these early variations of sO2-3D were correlated with fibrosis at 1 month (R=−0.48; p=0.04). Renal perfusion was altered in RIR-37°C at 20 minutes compared to basal values (median rBV 73 IQR [46-105] vs 100 IQR [89-126] a.u.; p=0.047), and remained altered at 1 month (median rBV 71 IQR [57-83] a.u.; p=0.03), but not in RIR-34°C (p=0.47 at both 20 minutes and 1 month). Renal perfusion early alterations at 20 minutes were correlated with late alterations at 1 month (R=0.63; p=0.005), and tended to correlate with fibrosis (R=−0.41; p=0.08). Conclusion Renal 3D-PAI (on the whole 3D-reconstructed kidney) and CEUS can detect early alterations of renal perfusion and oxygen saturation after RIR, and predict chronic disturbances of perfusion and the onset of fibrosis, as well as the protection conferred by mTH. 3D-PAI can also be used to evaluate non-invasively renal volume as a surrogate of renal fibrosis after RIR.

HSPA12A maintains aerobic glycolytic homeostasis and Histone3 lactylation in cardiomyocytes to attenuate myocardial ischemia/reperfusion injury.

Myocardial ischemia/reperfusion (MI/R) injury is a major cause of adverse outcomes of revascularization following myocardial infarction. Anaerobic glycolysis during myocardial ischemia is well studied, but the role of aerobic glycolysis during the early phase of reperfusion is incompletely understood. Lactylation of Histone H3 (H3) is an epigenetic indicator of the glycolytic switch. Heat shock protein A12A (HSPA12A) is an atypic member of the HSP70 family. In the present study, we report that, during reperfusion following myocardial ischemia, HSPA12A was downregulated and aerobic glycolytic flux was decreased in cardiomyocytes. Notably, HSPA12A KO in mice exacerbated MI/R-induced aerobic glycolysis decrease, cardiomyocyte death, and cardiac dysfunction. Gain- and loss-of-function studies demonstrated that HSPA12A was required to support cardiomyocyte survival upon hypoxia/reoxygenation (H/R) challenge and that its protective effects were mediated by maintaining aerobic glycolytic homeostasis for H3 lactylation. Further analyses revealed that HSPA12A increased Smurf1-mediated Hif1α protein stability, thus increasing glycolytic gene expression to maintain appropriate aerobic glycolytic activity to sustain H3 lactylation during reperfusion and, ultimately, improving cardiomyocyte survival to attenuate MI/R injury.

Remotely Triggered LAD Occlusion Using a Balloon Catheter in Spontaneously Breathing Mice.

Acute myocardial infarction (AMI) is a prevalent and high-mortality cardiovascular condition. Despite advancements in revascularization strategies for AMI, it frequently leads to myocardial ischemia-reperfusion injury (IRI), amplifying cardiac damage. Murine models serve as vital tools for investigating both acute injury and chronic myocardial remodeling in vivo. This study presents a unique closed-chest technique for remotely inducing myocardial IRI in mice, enabling the investigation of the very early phase of occlusion and reperfusion using in-vivo imaging such as MRI or PET. The protocol utilizes a remote occlusion method, allowing precise control over ischemia initiation after chest closure. It reduces surgical trauma, enables spontaneous breathing, and enhances experimental consistency. What sets this technique apart is its potential for simultaneous noninvasive imaging, including ultrasound and magnetic resonance imaging (MRI), during occlusion and reperfusion events. It offers a unique opportunity to analyze tissue responses in almost real-time, providing critical insights into processes during ischemia and reperfusion. Extensive systematic testing of this innovative approach was conducted, measuring cardiac necrosis markers for infarction, assessing the area at risk using contrast-enhanced MRI, and staining infarcts at the scar maturation stage. Through these investigations, emphasis was placed on the value of the proposed tool in advancing research approaches to myocardial ischemia-reperfusion injury and accelerating the development of targeted interventions. Preliminary findings demonstrating the feasibility of combining the proposed innovative experimental protocol with noninvasive imaging techniques are presented herein. These initial results highlight the benefit of utilizing the purpose-built animal cradle to remotely induce myocardial ischemia while simultaneously conducting MRI scans.

Oxidation–reduction imaging of myoglobin reveals two-phase oxidation in the reperfused myocardium

Myocardial infarction (MI) is a serious acute cardiovascular syndrome that causes myocardial injury due to blood flow obstruction to a specific myocardial area. Under ischemic–reperfusion settings, a burst of reactive oxygen species is generated, leading to redox imbalance that could be attributed to several molecules, including myoglobin. Myoglobin is dynamic and exhibits various oxidation–reduction states that have been an early subject of attention in the food industry, specifically for meat consumers. However, rarely if ever have the myoglobin optical properties been used to measure the severity of MI. In the current study, we develop a novel imaging pipeline that integrates tissue clearing, confocal and light sheet fluorescence microscopy, combined with imaging analysis, and processing tools to investigate and characterize the oxidation–reduction states of myoglobin in the ischemic area of the cleared myocardium post-MI. Using spectral imaging, we have characterized the endogenous fluorescence of the myocardium and demonstrated that it is partly composed by fluorescence of myoglobin. Under ischemia–reperfusion experimental settings, we report that the infarcted myocardium spectral signature is similar to that of oxidized myoglobin signal that peaks 3 h post-reperfusion and decreases with cardioprotection. The infarct size assessed by oxidation–reduction imaging at 3 h post-reperfusion was correlated to the one estimated with late gadolinium enhancement MRI at 24 h post-reperfusion. In conclusion, this original work suggests that the redox state of myoglobin can be used as a promising imaging biomarker for characterizing and estimating the size of the MI during early phases of reperfusion.

Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Myocardial oxidative stress is increased in early reperfusion, but systemic antioxidative therapy does not prevent ischemia-reperfusion arrhythmias in pigs.

Arrhythmias in the early phase of reperfusion after myocardial infarction (MI) are common, and can lead to hemodynamic instability or even cardiac arrest. Reactive oxygen species (ROS) are thought to play a key role in the underlying mechanisms, but evidence from large animal models is scarce, and effects of systemic antioxidative treatment remain contentious. MI was induced in 7 male and 7 female pigs (Norwegian landrace, 35-40 kg) by clamping of the left anterior descending artery (LAD) during open thorax surgery. Ischemia was maintained for 90 min, before observation for 1 h after reperfusion. Pigs were randomized 1:1 in an operator-blinded fashion to receive either i.v. N-acetylcysteine (NAC) from 70 min of ischemia and onwards, or 0.9% NaCl as a control. Blood samples and tissue biopsies were collected at baseline, 60 min of ischemia, and 5 and 60 min of reperfusion. ECG and invasive blood pressure were monitored throughout. The protocol was completed in 11 pigs. Oxidative stress, as indicated by immunoblotting for Malondialdehyde in myocardial biopsies, was increased at 5 min of reperfusion compared to baseline, but not at 60 min of reperfusion, and not reduced with NAC. We found no significant differences in circulating biomarkers of myocardial necrosis, nor in the incidence of idioventricular rhythm (IVR), non-sustained ventricular tachycardia (NSVT), ventricular tachycardia (VT) or ventricular fibrillation (VF) between NAC-treated and control pigs during reperfusion. Myocardial oxidation was increased early after reperfusion in a porcine model of MI, but systemic antioxidative treatment did not protect against reperfusion arrhythmias.

Neuroprotective effects of a hemoglobin-based oxygen carrier (stroma-free hemoglobin nanoparticle) on ischemia reperfusion injury

The application of hemoglobin (Hb)-based oxygen carriers (HBOCs) to the treatment of cerebral ischemia has been investigated. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA3) was found to exert neuroprotective effects on ischemia/reperfusion injury. Stroma-free hemoglobin nanoparticles (SFHbNP), a subsequently developed HBOC consisting of a spherical polymerized stroma-free Hb core with a HSA shell, contains the natural antioxidant enzyme catalase and, thus, is expected to exert additive effects. We herein investigated whether SFHbNP exerted enhanced neuroprotective effects in a rat transient middle cerebral artery occlusion (tMCAO) model. Rats were subjected to 2-hour tMCAO and divided into the following 3 groups with the intravenous administration of the respective reagents: (1) phosphate-buffered saline (PBS), as a vehicle (2) Hb-HSA3, and (3) SFHbNP. After 24-hour reperfusion, infarct and edema volumes decreased in the order of the PBS, Hb-HSA3, and SFHbNP groups, with a significant difference (p < 0.05) between the PBS and SFHbNP groups. Similar reductions were observed in oxidative stress, leukocyte recruitment, and blood–brain barrier disruption in the order of the PBS, Hb-HSA3, and SFHbNP groups. In the early phase of reperfusion within 6 h, microvascular HBOC perfusion and cerebral blood flow were maintained at high levels during the reperfusion period in the Hb-HSA3 and SFHbNP groups. However, a difference was observed in tissue oxygen partial pressure levels, which significantly decreased after 6-hour reperfusion in the Hb-HSA3 group, but remained high in the SFHbNP group. A superior oxygen transport ability appears to be related to the enhanced neuroprotective effects of SFHbNP.

Use of peroxiredoxin 6 to prevent liver dysfunction in acute kidney injury

Acute kidney injury causes deterioration of liver function, that is a confounding factor affecting treatment outcomes. In this work, renal ischemia reperfusion injury was used as a model. Taking into account that hyperproduction of reactive oxygen species is the major risk factor for kidney damage, the exogenous antioxidant enzyme peroxiredoxin 6, able to neutralize reactive oxygen species, has been used to prevent liver damage when kidneys are damaged. Kidney injury was initiated by a 45-minute ischemia simultaneously with a left-sided donor nephrectomy without manipulations of the liver. Peroxiredoxin 6 was administered intravenously 15 minutes before ischemia. The functional state of the liver was assessed after 2, 5 and 24 hours of reperfusion using histological and biochemical analysis. The signs of liver damage were detected in the best possible way after 5 hours of kidney reperfusion. It was found that peroxiredoxin 6 helps reduce the severity of the vascular reaction and leukocyte infiltration in the liver; lower the level of dystrophy and apoptosis of hepatocytes; keep the concentration of TBA-reactive products even and stabilize the level of cytokines, IL-6 and IL-10, in the liver tissue, as well as normalize the activity of intracellular transferases in the blood at the onset of reperfusion. The protective effect of peroxiredoxin 6 is associated primarily with its antioxidant properties, due to which hyperproduction of reactive oxygen species can be neutralized in the early phase of kidney reperfusion, but the signal-regulatory function of the protein can also contribute to a protective role peroxiredoxin 6.

The cardioprotective effects of pharmacological postconditioning with mangafodipir in ST-elevation myocardial infarction, a 4–7 years follow up study

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Futurum - the academy for health and care, Jönköping. Background/Introduction The prevalence of heart failure is increasing as more patients survive a myocardial infarction (MI). MI scar extent is a strong predictor of the development of heart failure due to left ventricular adverse remodeling. Reperfusion injury occurs in the early phase of reperfusion and is linked to the increase of reactive oxygen species (ROS) with calcium overload disrupting the mitochondrial structure, which in turn results in further cell necrosis. Manganese dipyridoxyl diphosphate (mangafodipir) has superoxide dismutase mimetics and may act as a ROS scavenger in the acute phase of myocardial ischemia, hence supporting the myocardial cells in the neutralization of the chemically reactive molecules reducing the MI extent [1–3]. Purpose Our aim was to investigate the cardioprotective role of pharmacological postconditioning with mangafidipir in patients with ST-elevation myocardial infarction (STEMI). Methods Twenty patients with acute STEMI were enrolled between 2009–2014 and followed up 2017. The subjects were randomized in the cath lab into blinded treatment with i.v mangafodipir or control treatment receiving i.v saline prior to restoration of blood flow in the occluded coronary artery. Cardiac magnetic resonance imaging (CMR) at baseline and at follow-up (4–7 years) was performed to evaluate MI scar, LV end diastolic volume (LVEDV), LV end systolic volume (LVESV), LV stroke volume (LVSV), LV ejection fraction (LVEF) and LV mass (LVM). The study was approved by the ethical review board in Linkoping (Dnr 2016/212-31) and registered in the public database at clinicaltrials.gov (NCT 00966563). Results There was no significant difference between the groups in MI scar or LV geometrical and functional properties at baseline, although the duration of ischemia was significantly longer in the mangafodipir group (206 min vs. 144 min, p &amp;lt; 0.05) and a better TIMI grade before pPCI was observed in the control group. The group that underwent pharmacological postconditioning with mangafodipir had lower volumes and higher LVEF (p &amp;lt; 0.05) at follow-up, while the placebo group showed increased LV volumes, see Table 1 and Figure 1. Conclusion Pharmacological postconditioning with mangafodipir in STEMI may have a cardioprotective role in the development of left ventricular adverse remodeling. Due to the limited number of participants, the benefit of mangafodipir needs confirmation in larger studies.

The neuroprotective role of prolonged normobaric oxygenation applied during ischemia and in the early stage of reperfusion in cerebral ischemic rats

BackgroundRecanalization is the main treatment option for ischemic stroke. However, prognosis remains poor for about half of patients after recanalization, possibly due to the “no-reflow” phenomenon at the early phase of recanalization. Normobaric oxygenation (NBO) during ischemia can reportedly maintain the partial pressure of oxygen and exert a protective effect in ischemic brain tissue. Objectives and methodsThis study investigated whether prolonged NBO treatment during ischemia and the early phase of reperfusion (i/rNBO) has neuroprotective effects and to elucidate the underlying mechanisms in rats with middle cerebral artery occlusion plus reperfusion. ResultsNBO treatment significantly elevated the level of O2 in the atmosphere and arterial blood without altering the level of CO2. The infarcted cerebral volume was significantly reduced by application of i/rNBO as compared to iNBO (applied during ischemia) or rNBO (applied at the early phase of reperfusion), indicating better protective effects of i/rNBO. i/rNBO more effectively suppressed s-nitrosylation of MMP-2 (amplifying inflammation) as compared to iNBO and rNBO, dramatically downregulated the cleavage of poly(ADP-ribose)polymerase-1 (PARP-1, acting as the substrate of MMP-2), and suppressed neuronal apoptosis, as determined by the TUNEL assay and staining for NeuN. These results demonstrated that application of i/rNBO in the early stage of reperfusion significantly alleviated neuronal apoptosis via suppression of the MMP-2/PARP-1 pathway. ConclusionsThe mechanism underlying the neuroprotective role of i/rNBO involved prolonged NBO treatment for cerebral ischemia, suggesting that i/rNBO may allow expansion of the time window for NBO application in stroke patients following vascular recanalization.

Characterization of early myocardial inflammation in ischemia-reperfusion injury.

Myocardial injury may be caused by myocardial ischemia-reperfusion (IR), and salvaging such an injury is still a great challenge in clinical practice. This study comprehensively characterized the physiopathologic changes of myocardial injury after IR to explore the underlying mechanism in the early reperfusion phase with particular emphasis on early myocardial inflammation. The experimental IR model was obtained by the left anterior descending artery's transient ligation of C57BL/6 mice. T2W signals of all mice showed increased signal at different IR stages. It was positively correlated with inflammatory cytokines and cells. T2W imaging by 7.0 T MRI surprisingly detected signal enhancement, but histopathology and flow cytometry did not reveal any inflammatory cells infiltration within 3 h after IR. Cardiomyocyte swelling and increased vascular permeability were observed by WGA staining and ultrastructural analysis, respectively. The 3 h IR group showed that the cardiomyocytes were severely affected with disintegrating myofilaments and mitochondria. Both VEGF and phosphorylated Src protein were markedly expressed in the 3 h IR group in comparison with the sham group, and TUNEL staining displayed little positive cells. Cleaved caspase-3 apoptin also has similar expression levels with that of the sham group. Resident macrophages had notably become M1 phenotype. The T2W signal was still elevated, and we observed that collagen deposition occurred from 1 to 7 days. The inflammation response during the first week after reperfusion injury gradually increase 3 h later, but the main manifestation before that was edema. This study indicated that the first 3 h may be crucial to the early rescue process for reperfusion-induced myocardial injury due to inflammatory cell infiltration absence and apoptosis.

Prospective analysis of serum albumin level as prognostic indicator in acute ischemic stroke

Background and Aim: Effect of albumin is primarily in the early reperfusion phase of acute ischemic stroke where it has an inhibitory effect on thrombosis, stagnation and adhesion of leucocytes in microcirculation. Aim of the study is to understand the association between serum albumin on admission and the functional status at 90 days. It also aims to find the other indicators that influence the outcome after ischemic stroke. Material and Methods: The study population consisted of total 200 patients who were admitted in the hospital wards with the instance of ischameic stroke within the first 2 hours of onset of symptoms. A detailed history was elicited from the attendees, followed by general examination. Serum albumin was measured using Bromocresol Green. Patients were followed up, and after 120 days following the onset of stroke, were evaluated either in person or over the phone using the Modified Ranking scale to assess their functional status. Results: Maximum numbers of patients were in severe disability score group. The prevalence of systemic hypertension was present tin 114 patients and absent in 86 patients. The association between GCS and albumin was found to be significant (p value<0.05) using the ANOVA test. The mean albumin in those with GCS>13 was 5.34 mg/dl while those for GCS< 9 was 4.26 mg/dl. There was a negative correlation between serum albumin at admission with the MRS score at 120 days. The higher the serum albumin levels, the lower the MRS score, hence better the outcome at 120 days. Conclusion: Serum albumin has a significant association with the severity as well as the prognosis of stroke.

Transferrin-mediated increase of labile iron Pool following simulated ischemia causes lipid peroxidation during the early phase of reperfusion

Heart ischemia/reperfusion (I/R) injury is related to iron content. However, the occurrence and mechanism of changes in labile iron pool (LIP) during I/R is debatable. Moreover, the identity of the iron form dominant in LIP during I/R is unclear. Herein, we measured changes of LIP during simulated ischemia (SI) and reperfusion (SR), in which ischemia was simulated in vitro with lactic acidosis and hypoxia. Total LIP did not change in lactic acidosis, whereas LIP, especially Fe3+, increased in hypoxia. Under SI, accompanied by hypoxia with acidosis, both Fe2+ and Fe3+ were significantly increased. Increased total LIP was maintained at 1 h post-SR. However, the Fe2+ and Fe3+ portion was changed. The increased Fe2+ was decreased, and conversely the Fe3+ was increased. BODIPY oxidized signal increased and through the time-course these changes correlated with blebbing of cell membrane and SR-induced LDH release. These data suggested lipid peroxidation occurred via Fenton’s reaction. The experiments using bafilomycin A1 and zinc protoporphyrin suggested no role of ferritinophagy or heme oxidation in the increase of LIP during SI. The extracellular source, transferrin assessed using serum transferrin bound iron (TBI) saturation showed that the depletion of TBI reduced SR-induced cell damages and additive saturation of TBI accelerated SR-induced lipid peroxidation. Furthermore, Apo-Tf dramatically blocked the increase of LIP and SR-induced damages. In conclusion, Tf-mediated iron induces the increase of LIP during SI, and it causes Fenton reaction-mediated lipid peroxidation during the early phase of SR.

Subacute Reperfusion in Ischemic Hearts: Study of Autophagy and its Possible Interconnection with Receptor-Interacting Protein Kinase 3.

The role of cardiac autophagy during ischemia and reperfusion (I/R) remains controversial. Furthermore, whether this cell death during I/R is also interconnected with other cell damaging event, such as necroptosis, is insufficiently known. Thus, the aim of this study was to investigate possible links between autophagy and necroptosis in the hearts under conditions of acute I/R injury. Langendorff-perfused male Wistar rat hearts were subjected to 30-min global ischemia followed by 10-min reperfusion in the presence of either vehicle or a drug inhibiting the pro-necroptotic receptor-interacting protein kinase 3 (RIP3). Hemodynamic parameters and lactate dehydrogenase (LDH) release were measured to assess heart function and non-specific cell death due to the disruption of plasma membrane. Immunoblot analysis of left ventricles revealed that early reperfusion suppressed the activation of autophagy as evidenced by the decreased protein expression of Beclin-1, pSer555-ULK1, pSer555-ULK1/ULK1 ratio, and LC3-II/LC3-I ratio. On the other hand, the molecular signalling responsible for autophagy inhibition did not appear to be affected in these I/R settings. RIP3 inhibition during reperfusion significantly mitigated the loss of the plasma membrane integrity but did not improve cardiac function. This pharmacological intervention targeting necroptosis-mediating protein decreased LC3-II expression in I/R hearts, suggesting some effect on autophagosome processing, but it did not significantly alter other signalling pathways involved in autophagy activation or inhibition. In summary, we showed for the first time that an early reperfusion phase does not promote autophagy and that there may be an interplay between pro-necroptotic protein RIP3 and autophagy with respect to the regulation of autophagosome processing.

A Novel Intraventricular Balloon Technique for Functional Assessment of Hearts from Donation After Circulatory Death

<h3>Purpose</h3> Functional assessment of hearts from donation after circulatory death (DCD) on ex-vivo heart perfusion (EVHP) is not well-established. Conventional intraventricular balloon (IVB) techniques, used for isolated heart perfusion in large animal models sacrifice the mitral valve to stabilize the balloon inside the left ventricle (LV), making it inapplicable for the clinical setting. This study demonstrates the effectiveness of a novel IVB technique to assess cardiac function during reperfusion and compares it to pressure-volume catheter measurements in a pediatric porcine DCD heart model. <h3>Methods</h3> Twelve pigs (9.5 ± 1.4 kg) were used. DCD hearts were harvested after 15 min of global warm ischemia due to asphyxic circulatory arrest and reperfused on EVHP for 2 hours. Isovolumetric LV pressure was measured during reperfusion with a saline-filled balloon inserted into the LV via a pulmonary vein orifice. The balloon was made from polyethylene wrap and connected to an 8 Fr sheath and pressure catheter, which was fixed with the left atrium via a purse-string suture around the origin of the pulmonary vein. DCD hearts were then switched to the working model (WM), pumping 120% of cardiac output, to measure cardiac function by using a pressure-volume catheter. Between-parameter correlations were assessed by Spearman's Rank Correlation Coefficient. <h3>Results</h3> Maximum first derivative of the LV pressure (dp/dt) measured by the IVB at 90 min of reperfusion showed moderate positive correlation (r_s = 0.61, p = 0.04) and coronary venous lactate levels showed moderate negative correlation (r_s = -0.55, p = 0.06) to respective measurements in the WM. Minimum dp/dt measured by the IVB at 60 min of reperfusion showed moderate positive correlation to measurements in the WM (r_s = 0.60, p = 0.04) and to tau in the WM (r_s = 0.52, p = 0.08). There was no significant difference between LV end-diastolic pressure during reperfusion and left atrial pressure in the WM. Myocardial oxygen consumption during reperfusion was less than 60% of that in the WM. <h3>Conclusion</h3> Functional parameters of contractility and relaxation measured by the IVB showed significant correlations to respective parameters in the WM. Therefore, this mitral valve sparing IVB technique can be used to confirm adequacy of cardiac function of DCD hearts in the early phase of reperfusion without the need to fully preload the heart.

3K3A-Activated Protein C Protects the Blood-Brain Barrier and Neurons From Accelerated Ischemic Injury Caused by Pericyte Deficiency in Mice.

Pericytes, mural cells of brain capillaries, maintain the blood-brain barrier (BBB), regulate cerebral blood flow (CBF), and protect neurons against ischemic damage. To further investigate the role of pericytes in ischemia, we induced stroke by 45-min transient middle cerebral artery occlusion (tMCAo) in 6-month-old pericyte-deficient Pdgfrb + ⁣/− mice and control Pdgfrb+/+ littermates. Compared to controls, Pdgfrb + ⁣/− mice showed a 26% greater loss of CBF during early reperfusion, and 40–50% increase in the infarct and edema volumes and motor neurological score 24 h after tMCAo. These changes were accompanied by 50% increase in both immunoglobulin G and fibrinogen pericapillary deposits in the ischemic cortex 8 h after tMCAo indicating an accelerated BBB breakdown, and 35 and 55% greater losses of pericyte coverage and number of degenerating neurons 24 h after tMCAo, respectively. Treatment of Pdgfrb + ⁣/− mice with 3K3A-activated protein C (APC), a cell-signaling analog of plasma protease APC, administered intravenously 10 min and 4 h after tMCAo normalized CBF during the early reperfusion phase and reduced infarct and edema volume and motor neurological score by 55–60%, with similar reductions in BBB breakdown and number of degenerating neurons. Our data suggest that pericyte deficiency results in greater brain injury, BBB breakdown, and neuronal degeneration in stroked mice and that 3K3A-APC protects the brain from accelerated injury caused by pericyte deficiency. These findings may have implications for treatment of ischemic brain injury in neurological conditions associated with pericyte loss such as those seen during normal aging and in neurodegenerative disorders such as Alzheimer’s disease.

Peripheral serotonin lacks effects on endothelial adhesion molecule expression in acute inflammation

BackgroundPeripheral, non‐neuronal serotonin promotes the recruitment of neutrophils to sites of acute inflammation and tissue damage. Direct effects of serotonin on neutrophil function were shown to be involved. However, the influence of serotonin on the endothelial counterpart is unknown. ObjectivesTo investigate whether serotonin alters the function of endothelial cells in leukocyte recruitment during acute inflammation. MethodsWe used two murine models of acute inflammation: intraperitoneal lipopolysaccharide (LPS) injection and mesenteric ischemia/reperfusion injury (I/R). To study effects of peripheral serotonin, leukocyte recruitment and endothelial adhesion molecule expression were compared in wild type (WT) and tryptophan hydroxylase 1 deficient (Tph1−/−) mice, which are unable to synthesize peripheral serotonin. ResultsAs expected, neutrophil transmigration into the peritoneal cavity following LPS injection was impaired in Tph1−/− mice. Abdominal blood vessels, however, showed no difference in adhesion molecule expression. In the early reperfusion phase after mesenteric I/R, the number of rolling leukocytes was significantly lower in Tph1−/− compared to WT. In line with the LPS model, endothelial adhesion molecule expression was independent of serotonin. In vitro experiments using human umbilical vein endothelial cells (HUVECs) confirmed that serotonin does not affect endothelial adhesion molecules. ConclusionsThe inflammatory release of peripheral serotonin is dispensable for the regulation of endothelial adhesion molecules.

Potentilla reptans L. postconditioning protects reperfusion injury via the RISK/SAFE pathways in an isolated rat heart

BackgroundOur previous study indicated that Potentilla reptans root has a preconditioning effect by its antioxidant and anti-apoptotic effects in an isolated rat heart ischemia/reperfusion (IR) model. In the present study, we investigated the post-conditioning cardio-protective effects of Potentilla reptans and its active substances.MethodsThe ethyl acetate fraction of P. reptans root (Et) was subjected to an IR model under 30 min of ischemia and 100 min of reperfusion. To investigate the postconditioning effect, Et was perfused for 15 min at the early phase of reperfusion. RISK/SAFE pathway inhibitors, 5HD and L-NAME, were applied individually 10 min before the ischemia, either alone or in combination with Et during the early reperfusion phase. The hemodynamic factors and ventricular arrhythmia were calculated during the reperfusion. Oxidative stress, apoptosis markers, GSK-3β and SGK1 proteins were assessed at the end of experiments.ResultsEt postconditioning (Etpost) significantly reduced the infarct size, arrhythmia score, ventricular fibrillation incidence, and enhanced the hemodynamic parameters by decreasing the MDA level and increasing expression of Nrf2, SOD and CAT activities. Meanwhile, Etpost increased the BCl-2/BAX ratio and decreased Caspase-3 expression. The cardioprotective effect of Etpost was abrogated by L-NAME, Wortmannin (a PI3K/Akt inhibitor), and AG490 (a JAK/STAT3 inhibitor). Finally, Etpost reduced the expression of GSK-3β and SGK1 proteins pertaining to the IR group.ConclusionP. reptans reveals the post-conditioning effects via the Nrf2 pathway, NO release, and the RISK/SAFE pathway. Also, Etpost decreased apoptotic indexes by inhibiting GSK-3β and SGK1 expressions. Hence, our data suggest that Etpost can be a suitable natural candidate to protect cardiomyocytes during reperfusion injury.

Abstract 11558: The Role of Oxidized CaMKII in Early Ischemia-Reperfusion Triggered Ventricular Arrhythmias

Introduction: Arrhythmias can complicate revascularization procedures after transmural infarctions and induce hemodynamic instability, but most cases can be adequately treated when they occur in hospital. However, arrhythmias that occur outside of hospital are potentially fatal. Arrhythmias in the early phase of reperfusion are caused by Ca 2+ overload and spontaneous diastolic release of Ca 2+ from the sarcoplasmic reticulum (SR). Indirect evidence suggests that reactive oxygen species (ROS) are important in this process by affecting the activity of key Ca 2+ handling proteins. Specifically, previous publications propose oxidation of the multifunctional Ca 2+ /calmodulin dependent protein kinase type II (CaMKII) to be a critical factor, and a potential therapeutic target. There is, however, a lack of data providing direct evidence for the importance of oxidized CaMKII (ox-CaMKII) for triggered arrhythmias in the early phase of reperfusion. Hypothesis: We hypothesized that ox-CaMKII increases diastolic SR Ca 2+ release events in the early phase of reperfusion. Methods: We evaluated the propensity for early reperfusion arrhythmias in mice resistant to oxidation of CaMKII in the heart (homozygous CaMKIIδ M281/282V mice) and wild type control mice (WT), by the use of Langendorff-perfused hearts exposed to ischemia-reperfusion, confocal line scan imaging of isolated cardiomyocytes transiently exposed to hypoxia and ischemia-like conditions, and immunoblotting. Results: Langendorff-perfused hearts from CaMKIIδ M281/282V mice showed no significant difference in ventricular arrhythmias compared to WT during the early reperfusion phase, both at baseline and during beta-adrenoceptor stimulation with isoprenaline. Immunoblotting of left ventricular tissue from perfused hearts revealed no significant differences in the abundance or phosphorylation levels of Ca 2+ -handling proteins. Ca 2+ imaging experiments without isoprenaline showed no significant difference in the incidence of spontaneous transients, Ca 2+ waves or Ca 2+ sparks between CaMKIIδ M281/282V and WT mice. Conclusions: Initial data indicate that ablation of CaMKII oxidation does not protect mouse hearts against arrhythmias in the early phase of reperfusion after ischemia.