Reperfusion Period Research Articles

Functional, Metabolic and Morphologic Results of Ex Vivo Donor Lung Perfusion with a Perfluorocarbon-Based Oxygen Carrier Nanoemulsion in a Large Animal Transplantation Model.

Background: Ex vivo lung perfusion (EVLP) is a technology that allows the re-evaluation of questionable donor lung before implantation and it has the potential to repair injured donor lungs that are otherwise unsuitable for transplantation. We hypothesized that perfluorocarbon-based oxygen carrier, a novel reconditioning strategy instilled during EVLP would improve graft function. Methods: We utilized perfluorocarbon-based oxygen carrier (PFCOC) during EVLP to recondition and improve lung graft function in a pig model of EVLP and lung transplantation. Lungs were retrieved and stored for 24 h at 4 °C. EVLP was done for 6 h with or without PFCOC. In the transplantation groups, left lung transplantation was done after EVLP with or without PFCOC. Allograft function was assessed by means of pulmonary gas exchange, lung mechanics and vascular pressures, histology and transmission electron microscopy (TEM). Results: In the EVLP only groups, physiological and biochemical markers during the 6-h perfusion period were comparable. However, perfusate lactate potassium levels were lower and ATP levels were higher in the PFCOC group. Radiologic assessment revealed significantly more lung infiltrates in the controls than in the PFCOC group (p = 0.04). In transplantation groups, perfusate glucose consumption was higher in the control group. Lactate levels were significantly lower in the PFCOC group (p = 0.02). Perfusate flavin mononucleotide (FMN) was significantly higher in the controls (p = 0.008). Post-transplant gas exchange was significantly better during the 4-h reperfusion period in the PFCOC group (p = 0.01). Plasma IL-8 and IL-12 levels were significantly lower in the PFCOC group (p = 0.01, p = 0.03, respectively). ATP lung tissue levels at the end of the transplantation were higher and myeloperoxidase (MPO) levels in lung tissue were lower in the PFCOC group compared to the control group. In the PFCOC group, TEM showed better tissue preservation and cellular viability. Conclusion: PFCOC application is safe during EVLP in lungs preserved 24 h at 4 °C. Although this strategy did not significantly affect the EVLP physiology, metabolic markers of the donor quality such as lactate production, glucose consumption, neutrophil infiltration and preservation of mitochondrial function were better in the PFCOC group. Following transplantation, PFCOC resulted in better graft function and TEM showed better tissue preservation, cellular viability and improved gas transport.

Perioperative Cardioprotection: General Mechanisms and Pharmacological Approaches.

Cardioprotection encompasses a variety of strategies protecting the heart against myocardial injury that occurs during and after inadequate blood supply to the heart during myocardial infarction. While restoring reperfusion is crucial for salvaging myocardium from further damage, paradoxically, it itself accounts for additional cell death-a phenomenon named ischemia/reperfusion injury. Therefore, therapeutic strategies are necessary to render the heart protected against myocardial infarction. Ischemic pre- and postconditioning, by short periods of sublethal cardiac ischemia and reperfusion, are still the strongest mechanisms to achieve cardioprotection. However, it is highly impractical and far too invasive for clinical use. Fortunately, it can be mimicked pharmacologically, for example, by volatile anesthetics, noble gases, opioids, propofol, dexmedetomidine, and phosphodiesterase inhibitors. These substances are all routinely used in the clinical setting and seem promising candidates for successful translation of cardioprotection from experimental protocols to clinical trials. This review presents the fundamental mechanisms of conditioning strategies and provides an overview of the most recent and relevant findings on different concepts achieving cardioprotection in the experimental setting, specifically emphasizing pharmacological approaches in the perioperative context.

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons.

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-KATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-KATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15s reperfusion and 15s re-ischemia, and was started 30s after the 7.5min ischemic load. We recorded N-methyl-D-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca2+ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-KATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca2+ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca2+ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-KATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

От «пустой» липосомы до сложной наноструктуры

В работе подводятся некоторые итоги почти 40-летней работы авторов в области липосомологии, начатые в 80-х годах прошлого века под руководством академика НАМН Украины Александра Викторовича Стефанова.Создание нужной концентрации лекарственных веществ в органе-мишени, не затрагивая при этом не вовлеченные в патологический процесс органы и ткани, – задача непростая. За последние несколько лет предпринято множество попыток решить эту проблему, и оказалось, что лучшимипереносчиками лекарств являются именно липосомы.Преимущества липосом перед другими носителями обусловлены такими их свойствами:– сродство с природными мембранами клеток по химическому составу;– универсальность. Липосомы способны переносить разнообразные фармакологически активныевещества, а также дополнительные источники энергии для клетки и генетический материал; 
 - липосомы сравнительно легко биодеградируют, высвобождая доставленные вещества. В пути следования липосомы надежно укрывают свое содержимое от контакта с иммунной системой и не вызывают защитных и аллергических реакций организма;– способ взаимодействия липосом с клетками, который происходит в разных формах. При этом могут изменяться свойства клеточных мембран, например: вязкость и проницаемость; величина электрического заряда; фосфолипидное окружение ферментов и ионных каналов и, соответственно, их активность (вот почему даже так называемая пустая липосома может быть биологически активным агентом).Благодаря липосомам появляется новый способ направленного воздействия на клетку, который можно назвать «мембранной инженерией», то есть, появляется возможность модифицировать клеточную мембрану в заданном направлении.Оказалось, что липосомы могут быть не только переносчиками лекарств, но и обладают самостоятельной биологической активностью. Продемонстрирована способность так называемых пустых фосфатидилхолиновых липосом (ФХЛ) оказывать влияние на электрическую и сократительную активность сосудистых гладких мышц. При гипоксии ФХЛ восстанавливают измененную для ионов проницаемость плазматических мембран гладких мышц сосудов, а у крыс со спонтанной артериальной гипертензией улучшают эндотелий-зависимые дилататорные реакции сосудов.Кроме того, было показано, что «пустые» ФХЛ эффективно восстанавливают вызванное ацетилхолином эндотелий-зависимое расслабление изолированных колец грудной аорты, нарушенное воздействием гамма-облучения. Липосомы также восстанавливают пониженную после облучения чувствительность сосудистой ткани к ацетилхолину и оксиду азота.С целью усиления антигипоксического и антиоксидантного действия липосом в их структуру был внедрен антиоксидант кверцетин. Использование липосомальной формы кверцетина приводит к полноценному восстановлению коронарного кровотока в послеокклюзионном периоде приострой субтотальной ишемии миокарда, в значительной степени уменьшает проявления характерного для репеперфузионного периода окислительного стресса и предупреждает повышение содержания лейкотриенов С4 и Е4. Синтез простациклина при этом не уменьшается. Применение липосомальной формы кверцетина приводит к уменьшению зоны некроза миокарда в два раза.Липосомальная форма кверцетина практически полностью предотвращает или в значительной степени корригирует нарушения ритма сердечных сокращений и восстанавливает сократительную активность миокарда.Липосомальная форма кверцетина эффективно восстанавливает функцию калиевых каналов большой проводимости у облученных крыс.Перспективным может оказаться включение в состав липосом цитохрома С. Липосомальная форма цитохрома С приводит к коррекции нарушений гемостаза и предотвращает развитие синдрома ДВС при острой массивной кровопотере.Многочисленные патологические процессы, такие как гипертония, атеросклероз, нарушение кровотока в центральной нервной системе, вызваны недостаточностью эндогенного синтеза/высвобождения NO. В таких случаях дополнительное введение NO может быть очень эффективнойстратегией в лечении заболеваний, связанных с указанными патологическими процессами.Разработка современных доноров оксида азота, обладающих способностью контролируемого и дозированного высвобождения действующего вещества, представляет собой чрезвычайно сложную задачу. Создание липидной системы доставки NO, способной обеспечить естественную скорость высвобождения, и разработка ее стабильной лекарственной формы стало следующей целью наших исследований.Проведенные исследования специфического действия сконструированной липосомальной формы оксида азота (ЛФ NO) свидетельствуют о его ее чрезвычайно высокой дилататорной, антиишемической активности. Это подтверждается выраженным дозозависимым расслаблением сегментов интактной аорты крыс и продолжительной гипотензивной реакцией системного кровообращения. ЛФ NO предотвращает развитие критических нарушений насосной функции сердца и способствует сохранению его сократительной активности при субтотальной ишемии. Действие ЛФ NOпроявляется значительным также дозозависимым расслаблением полосок пещеристых тел полового члена и миометрия.Новый NO-содержащий липосомальный конструкт эффективно открывает одиночные кальцийзависимые калиевые каналы большой проводимости в сосудистых гладких мышцах и значительно увеличивает вероятность их нахождения в открытом состоянии.Таким образом, ЛФ NO обещает быть эффективной при лечении ангиопатий различного генеза (включая диабетическую), эректильной дисфункции, гиперактивного мочевого пузыря, артериальной гипертензии, геморрагического и ишемического инсульта. Учитывая возможность применения в ингаляционной форме, она может быть использована для лечения легочной гипертензии.Высказывается мнение, что потенциальные возможности липосом существенно выше пока известных нам. Мы только начали понимать, как липосомы работают с клеткой.

Cardioprotection by remote ischemic conditioning is transferable by plasma and mediated by exosomes

Abstract Background Remote ischemic conditioning (RIC) by brief periods of limb ischemia and reperfusion protects against ischemia-reperfusion injury. However, the mechanism is unknown. Purpose We studied the role of exosomes for mediating the cardioprotective signal and whether they accumulate in injured myocardium. Methods Blood samples from 12 healthy male volunteers were obtained prior to and one hour after RIC. Plasma obtained before and after RIC (n=4) (P-Pre and P-Post) was used to evaluate the transferability of RIC. Pre- and Post-RIC plasma (n=8) was separated into an exosome rich fraction (Exo-Pre and Exo-Post) and an exosome depleted fraction (Prot-Pre and Prot-Post) by size exclusion chromatography. All studies were carried out in duplicate samples from each volunteer. Infarct size was compared in Sprague-Dawley rat hearts perfused with plasma, exosomes and exosome depleted fractions in a Langendorff model. We investigated changes in the miRNA content of the exosomes after RIC by a human miRNA panel. Additionally, fluorescently labeled exosomes isolated from C2C12 cells were used to assess accumulation in injured myocardium in an in vivo rat model. Rats were divided into an infarct group (n=6) (left anterior descending artery ligation) and a sham group (n=6) (without ligation). Labelled exosomes were injected in the femoral vein prior to reperfusion. Exosome-accumulation in infarcted or sham myocardium was evaluated. Results P-Post reduced infarct size by 15% points compared with P-Pre (55±4% vs 70±6%, p=0.03) (Fig. 1a). Exo-Post reduced infarct size by 16% points compared with Exo-Pre (53±15% vs 68±12%, p=0.03) (Fig. 1b). Prot-Post did not affect infarct size compared to Prot-Pre (64±3% and 68±10%, p>0.99). We found miRNA-16, miRNA-144 and miRNA-451 to be upregulated in exosomes after RIC and the mTOR-pathway as a potential target for these miRNAs. In the in vivo model, labelled exosomes accumulated more intensively in the infarct area than in remote areas and sham hearts (Fig. 1c). Conclusion Cardioprotection by RIC is mediated by exosomes with a changed miRNA profile and exosomes accumulate in injured myocardium. Figure 1 Funding Acknowledgement Type of funding source: Private company. Main funding source(s): Novo synergy

In Vitro Oxygen-Glucose Deprivation-Induced Stroke Models with Human Neuroblastoma Cell- and Induced Pluripotent Stem Cell-Derived Neurons.

Stroke is a devastating neurological disorder and one of the leading causes of mortality and disability. To understand the cellular and molecular mechanisms of stroke and to develop novel therapeutic approaches, two different in vitro human cell-based stroke models were established using oxygen-glucose deprivation (OGD) conditions. In addition, the effect of adipose stem cells (ASCs) on OGD-induced injury was studied. In the present study, SH-SY5Y human neuroblastoma cells and human induced pluripotent stem cells (hiPSCs) were differentiated into neurons, cultured under OGD conditions (1% O2) for 24 h, and subjected to a reperfusion period for 24 or 72 h. After OGD, ASCs were cocultured with neurons on inserts for 24 or 72 h to study the neuroprotective potential of ASCs. The effect of OGD and ASC coculture on the viability, apoptosis, and proliferation of and axonal damage to neuronal cells was studied. The results showed that OGD conditions induced cytotoxicity and apoptosis of SH-SY5Y- and hiPSC-derived neurons, although more severe damage was detected in SH-SY5Y-derived neurons than in hiPSC-derived neurons. Coculture with ASCs was protective for neurons, as the number of dead ASC-cocultured neurons was lower than that of control cells, and coculture increased the proliferation of both cell types. To conclude, we developed in vitro human cell-based stroke models in SH-SY5Y- and hiPSC-derived neurons. This was the first time hiPSCs were used to model stroke in vitro. Since OGD had different effects on the studied cell types, this study highlights the importance of using several cell types in in vitro studies to confirm the outcomes of the study. Here, ASCs exerted a neuroprotective effect by increasing the proliferation and decreasing the death of SH-SY5Y- and hiPSC-derived neurons after OGD.

Effects of Hesperetin on the Serum Superoxide Dismutase, Glutathione Peroxidase, and Malondialdehyde Levels and Renal-Histopathological Alterations Following Skeletal Muscle Ischemia Reperfusion in Male Rats

Objectives: The aim of the current study was to assess the effects of hesperetin on damage to kidneys as remote organs following skeletal muscle ischemia-reperfusion (IR) in rats. Materials and Methods: In general, 30 male Wistar rats were randomized and placed into sham, IR, hesperetin, dimethyl sulfoxide (DMSO), and IR+hesperetin groups. The rats in the hesperetin and IR+hesperetin groups received a 50 mg/kg dose of hesperetin dissolved in DMSO intraperitoneally. In the IR+hesperetin group, hesperetin was injected exactly prior to reperfusion. To induce skeletal muscle ischemia, the femoral artery was clamped for two hours. Following a 24-hour period of reperfusion, the samples of blood were collected for renal function tests and oxidative stress measurements. Next, the rats were euthanized, and histological analyses were conducted on their removed kidneys. Results: Based on the results, urea and creatinine serum levels were significantly higher in the IR group (P<0.05) whereas they significantly reduced following treatment with hesperetin (P<0.05). The concentration of malondialdehyde (P<0.05) increased for the IR group while those of superoxide dismutase (P<0.05) and glutathione peroxidase (P<0.05) activities were lower than the other groups. The analysis of renal tissues in the IR group showed glomerular necrosis, degeneration, and necrosis of the tubular epithelium, protein casts, interstitial edema, and inflammation. Finally, the degree of renal injury was significantly ameliorated (P<0.05) in rats treated with hesperetin. Conclusions: Overall, the results indicated that in rats, hesperetin could reduce renal injury that has been induced through skeletal muscle IR.

Evaluating the possible role of mitochondrial ATP-sensitive potassium channels in the cardioprotective effects of morin in the isolated rat heart

AimsDuring heart ischemia, the lack of oxygen in the myocardial cells causes pH and ion disturbances and cell death through opening mitochondrial permeability transition pores (mPTP). Considering the inhibitory effects of mitochondrial ATP-dependent potassium channels (mt-KATP) on these pores and anti-ischemic effects of morin, we hypothesized that it may exert its positive effects via activating mt-KATP as well as its anti-oxidative effects. Main methodsIsolated rat hearts were perfused by Krebs-Henseleit solution enriched with the morin (0.25, 0.5 and 1 mg/L) or 5-hydroxydecanoate (5-HD, a mt-KATP blocker;100 μM) or both as needed 5 min before starting regional ischemia till the first 10 min of the reperfusion period. The reperfusion was developed with Krebs-Henseleit solution 60 or 120 min respectively for biochemical evaluations (lactate dehydrogenase and malondialdehyde level) or the assessment of myocardial infarct size. During the experiments, hemodynamic functions were recorded and cardiac arrhythmias were determined. Key findingsOur findings demonstrated that morin reduced the infarct size. Also, morin perfusion could remarkably prevent the malondialdehyde over-production during ischemia. Total ventricular ectopic beats had the same significant changes as the malondialdehyde level, in both ischemia and reperfusion phases. Morin could also relatively improve the ischemia-induced hemodynamic dysfunction. All mentioned protective effects of morin were reversed by concomitant perfusion of 5-HD. SignificanceMorin has protective effects against ischemic hearts through anti-oxidative effects. It also suggests a link between the cardioprotective effects of morin and mt-KATP. However, additional studies are required to prove this preliminary hypothesis.

The study of myocardial ischemia-reperfusion treatment through computational modelling

Reperfusion of the blood flow to ischemic myocardium is the standard treatment for patients suffering myocardial infarction. However, the reperfusion itself can also induce myocardial injury, in which the actual mechanism and its risk factors remain unclear. This work aims to study the mechanism of ischemia–reperfusion treatment using a three-dimensional (3D) oxygen diffusion model. An electrical model is then coupled to an oxygen model to identify the possible region of myocardial damage. Our findings show that the value of oxygen exceeds its optimum (>1.0) at the ischemic area during early reperfusion period. This complication was exacerbated in a longer ischemic period. While a longer reperfusion time causes a continuous excessive oxygen supply to the ischemic area throughout the reperfusion time. This work also suggests the use of less than 0.8 of initial oxygen concentration in the reperfusion treatment to prevent undesired upsurge at the early reperfusion period and further myocardial injury. We also found the region at risk for myocardial injury is confined in the ischemic vicinity revealed by its electrical conductivity impairment. Although there is a risk that reperfusion leads to myocardial injury for excessive oxygen accumulation, the reperfusion treatment is helpful in reducing the infarct size.

Changes of biochemical indicators of blood serum and the skeletal muscle homogenate of the hind limbs of the rats in acute ischemia-reperfusion and in the condition of using of the carbacetam

Annotation. Simulation of ischemia-reperfusion causes the biochemical disorders and changes in the prooxidant-antioxidant system, both in the serum and skeletal muscles of the affected limb. The severity of local and systemic postischemic imbalance decreases in with correction with medicine from the group of nootropics – carabacetam. The aim of the study was determining the degree of metabolic disorders based on the analysis of changes in serum biochemical parameters and muscle homogenate in ischemic-reperfusion disorders caused by the imposition of an arterial tourniquet and under conditions of correction with carbacetam. Materials and methods. A biochemical study of blood serum and muscle homogenate of 60 rats was performed. Ischemia was simulated by applying SWAT rubber tourniquets on the hind limbs of the rats, on the inguinal fold, for 2 hours, and reperfusion was modeled by removing the tourniquet and restoring of blood circulation through 2 hours after its application. Observations of the animals were carried out during for 14 days. 30 rats in the reperfusion period were intraperitoneally administered the carbacetam at a dose of 5 mg per kilogram of body weight 1 time a day for 14 days of the reperfusion period. The Blood serum was separated and the soft tissue samples were taken below the turniquet site for the biochemical studies. Statistical processing of the material was performed using the package of programs “Microsoft Excel”. The largest deviations in serum were observed after 1 day of the reperfusion: an increase in creatinine by 28.16 % (p<0.005), total bilirubin by 20.75 % (p<0,005), ALT by 96.14 % (p<0,005), AST by 147.27 % (p<0.005) and alkaline phosphatase by 70.84 % (p<0.005), as well as an increase in prooxidant activity of CD by 77.27 % (p<0.005), CT 147.47 % (p<0,005) and TBC-AL by 82.52 % (p<0,005) against the background of a decrease in SOD activity by 9.77 % (p<0.005) and an increase in catalase levels by 15.37 % (p<0.005) relative to the value of the control group. The development of ischemia and reperfusion was accompanied by the largest increase in the level of prooxidants (CD – by 27.55 % (p<0.005), CT – by 47.34 % (p<0.05), TBC-AL – by 62.09 %) (p<0.005)) in the homogenate of the skeletal muscles after 2 hours after restoration of the blood supply. Antioxidant activity, on the other hand, decreased by 15.06 % (p<0.005) SOD and by 8.96 % (p<0.005) catalase after 1 day. After 14 days, a gradual increase and return of most of the studied values to the level of the control group. Under the influence of the carbacetam, the biochemical parameters of blood serum most significantly decreased in relative to the value of the groups without correction after 7 days of the experiment. The level of total bilirubin was lower by 29.65 % (p˂0.05), ALT by 14.95 % (p˂0.05), AST by 19.84 % (p˂0.05), alkaline phosphatase by 25.53 % (p˂0.05), and the difference in the content of diene, triene conjugates and TBC-LP was the largest after 1 day – by 17.09 % (p<0.005), 20,41 % (p<0.05) and 11.24 % (p<0.05), respectively. Under the conditions of correction, the indicators of SOD and catalase activity increased, being significantly higher than in the groups of the animals without correction, especially after 7 days of reperfusion – by 29.41 % (p<0.005) and by 15.71 % (p<0.05) in accordance. The content of the diene, triene conjugates and TBC-LP muscle homogenate was statistically significantly lower in the groups with the introduction of the carbacetam than in the groups of the animals without correction. The largest difference between the groups was found after 1 day with a difference of 9.16 % (p<0.05), 7.35 % (p<0.005) and 16.05 % (p<0.05), respectively. Antioxidant activity of SOD and catalase after 1 day was higher by 27.41 % (p<0.005) and by 25.13 % (p<0.005), compared with the group of the animals without introduction of the carbacetam. Thus, simulated ischemia-reperfusion is accompanied by the greatest biochemical disorders in the serum – after 1 day, and in the muscle homogenate – after 2 hours of the reperfusion. After 14 days of the experiment, most indicators still do not return to the level of the control group. The application of the correction helps to reduce the severity of biochemical disorders at the systemic and local levels, especially during periods of their greatest deviations.

Histological changes of the arterial bed of the hind limbs of the rats under condition of the acute ischemia-reperfusion and correction with the carbacetam

The ischemic-reperfusion lesion is a complex multifactorial damage of the primary ischemic tissues as a result of restoration of the arterial blood circulation in them, which is accompanied by local morpho-functional reorganization of the vascular bed of the hind limbs of the rats. One of the promising means in the treatment and prevention of the reperfusion disorders is a carbacetam, which smooths the phenomena of hypo- and hyperperfusion in the post-ischemic period. The aim of the study was to established the manifestations of the morpho-functional remodeling of the vascular bed of the hind limbs of the rats in ischemia-reperfusion and under conditions of correction with carbacetam. Histological examination of the vascular bed of the hind limbs of 30 rats under conditions of ischemia-reperfusion (group I) and 30 rats in the simulation of ischemia-reperfusion, which in the post-ischemic period administered carbacetam once a day (5 mg/kg) for 14 days (group II) were done. There were 6 intact animals in the control group. Simulation of ischemia was performed by applying SWAT rubber tourniquets on the hind limbs for 2 hours, and reperfusion – by removing of the tourniquet. The animals of the experimental groups were divided into 5 subgroups with reperfusion terms after 1, 2 hours and 1 day, as well as after 7 and 14 days. Histological examination was performed according to generally accepted methods. The vascular bed in the middle third of the thigh and the shin below the tourniquet was examined using a Bresser Trino Researcher 40x–1000x microscope. Analyzing of the obtained results, was established that after 1 hour of the reperfusion the histological changes became a systemic, and after 1 day it were more significant. It should be noted that the thickness of the vessel walls increased, and the elastic membranes were partially eligned, thinned and torned. The stepwise clarity of the arterials walls structure was lost. The edema acquired a total nature. The histological examination of the vessels after 7 days revealed that the swelling of the walls decreased and the condition of the elastic frame was improved. There was a proliferation of collagen fibers in the adventitia, which was a response to ischemic effects. It is noted that after 14 days in all wall membranes the proliferative activity of fiboblasts was remained. Under the conditions of the correction with the carbacetam after 2 hours, the structural positive dynamics became more pronounced and increased to a maximum level after 7 days of the experiment. The number of the modified and exfoliated endothelial cells decreased, and the condition of smooth myocytes increased. Histologically, the gradual restoration of endothelial coverage of the intima was established. As follows, ischemia and reperfusion cause vascular remodeling after 1 hour with a peak of the manifestations after 1 day of the reperfusion, which includes edematous syndrome, dystrophic-degenerative changes with an inflammatory response to the damage, and in the late reperfusion period increased a fibroblasts activity. Gradual return of morphological changes occurs after 14 days of the experiment. Under the conditions of correction, the acceleration of the remodeling with stabilization of the process and the most possible structural restoration after 7 days of the study was noted.

Changes in the blood coagulation system and non-specific plasma proteinases in ischemia-reperfusion injury

The aim of this study was to determine the general patterns of pathogenetic changes in the blood coagulation system and in non-specific proteinases and their inhibitors during the development of experimental ischemiareperfusion injury. Materials and methods. The study was conducted on 48 male Wistar rats (180–200 g). We used a model of ischemia-reperfusion injury achieved by applying rubber tourniquets to both hind limbs at the inguinal fold level for 6 hours. Revascularization was performed for 6, 12, or 24 hours following the application of tourniquets, after which we examined the state of the internal and external blood coagulation pathways and the activity of nonspecific proteinases and their inhibitors. Results. Indicators of blood coagulation system change show the development of blood hypocoagulation changes as the reperfusion time increases. By the 6th hour of reperfusion, the prothrombin time (PT) was lengthened by 112.0% ( p = 0.0142) and the activated partial thromboplastin time (APTT) by 170.0% ( p = 0.0147) compared with values in the control group. By the 12th reperfusion hour, the PT was lengthened by 174.2% ( p = 0.0389), and the APTT increased 4.9-fold ( p = 0.0002). When the reperfusion period was increased to 24 hours, it was characterized by lengthened PT and APTT, accompanied by an increase in antithrombin III by 11.5% ( p = 0.0371) and a decrease in protein C by 71.4% ( p = 0.0071). Changes in the non-specific proteinases and their inhibitors were characterized by a 2.8-fold increase in the trypsin-like proteinase activity ( p < 0.001) relative to the control, as well as a 2.2-fold decrease in antitrypsin activity and acid-stable inhibitors ( p < 0.001), which reached a maximum after 24 hours of reperfusion. A direct correlation was found between indicators characterizing the deficiency of coagulation system factors and a decrease in antiproteinase potential. Conclusion. Hemostatic system disorders are characterized by the development of hypocoagulation during ischemia-reperfusion injury as the result of an increase in the trypsin-like proteinase activity and a decrease in the levels of inhibitors. The established changes may be associated with the deficiency of coagulation factors and proteinase inhibitors and share common pathogenic mechanisms.

IMPROVED ACUTE ST-SEGMENT ELEVATION DUE TO AIR EMBOLISM AFTER CORONARY ARTERY BYPASS GRAFT PROCEDURE

SESSION TITLE: Medical Student/Resident Cardiovascular Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Air embolism is often an iatrogenic complication during coronary artery bypass grafting (CABG) surgery.1 Although infrequent as a complication of CABG surgery it is generally attributed to air introduced into the coronary arteries during the procedure.2 We report a case of ST-segment elevation after triple CABG surgery secondary to the introduction of air into the coronary arteries. CASE PRESENTATION: A 62-year-old male presents to the emergency department with mid-sternal chest pain, radiating to the left arm, his symptoms were associated with diaphoresis and palpitations. Initial Electrocardiogram (ECG) showed lateral ST-segment depressions, the initial troponin was elevated at 0.32 ng/ml (0.015-0.045 ng/ml). Patient underwent a left heart catheterization, which showed severe multi-vessel coronary artery disease. The decision was made to proceed with CABG. CT surgery was consulted and he underwent triple CABG with left internal mammary artery grafting to the anterior descending coronary artery, saphenous venous grafting to obtuse marginal 1 branch and saphenous venous grafting to the posterolateral branch of the right coronary artery. The anastomosis was completed with successful filling of the grafts. The aortic cross-clamp was removed, and the patient was allowed adequately to reperfuse and rewarm. Shortly after that, the patient developed ST-segment elevation in lead II, III, and aVF, along with ST depressions in lead I, aVL, V1, V2. Flow in the grafts were checked and they remained patent. Air embolization of the coronary arteries was suspected. Transthoracic echocardiogram (ECHO) demonstrated ventricular function impairment. Extended reperfusion was performed to force air from the coronaries out. The ECG showed resolution of ST-segment elevations and repeat ECHO showed improved ventricular function. The patient was monitored in the cardiovascular intensive care unit and was discharged home after four days of routine Post-CABG care. DISCUSSION: Air embolization of the coronary arteries may result as a complication of CABG as well as cardiac catheterization, leading to disruption of coronary perfusion.1 There may also be elevations of cardiac enzymes and hemodynamic instability. An extended reperfusion period with increased perfusion pressures usually causes the passing of bubbles through the coronary artery system, during the surgery.3,4 Late embolizations require urgent re-catheterization. 4 CONCLUSIONS: In conclusion, this case illustrates ST-segment elevation on ECG from an air embolism during CABG surgery. Early diagnosis and prompt treatment of this rare iatrogenic complication are crucial to prevent life-threatening cardiac complications. Reference #1: 1. Engelmann U, Minden H, Stock UA, Kühnel R, Albes JM. Late air embolism with interventional removal after isolated coronary artery bypass grafting. J Thorac Cardiovasc Surg. 2006;131(6):1403-1404.doi:10.1016/j.jtcvs.2006.01.046 2. Khan M, Schmidt DH, Bajwa T, Shalev Y. Coronary air embolism: Incidence, severity, and suggested approaches to treatment. Cathet Cardiovasc Diagn. 1995;36(4):313-318. doi:10.1002/ccd.1810360406 3. Solodky A, Birnbaum Y, Assali A, Gal T Ben. Coronary Air Embolism Treated by Bubble Aspiration. 2000;454(October 1999):452-454. 4. Patterson MS, Kiemeneij F. Coronary air embolism treated with aspiration catheter. Heart. 2005;91(5):1-2. doi:10.1136/hrt.2005.060129 Reference #2: Figure 1. Initial Electrocardiogram. Figure 2. Post CABG Electrocardiogram. Figure 3. Electrocardiogram at discharge. DISCLOSURES: No relevant relationships by Ahmad ababneh, source=Web Response No relevant relationships by Shubham Adroja, source=Web Response No relevant relationships by HANAD BASHIR, source=Web Response No relevant relationships by Ahmad JABRI, source=Web Response

Structural changes in skeletal muscles of the hind limbs of rats in acute ischemia-reperfusion and its correction by carbacetam, detected by polarization microscopy

Arterial tourniquets are used in clinical practice for angioplasty and arthroplasty, and in case of limb injuries, their use often occurs according to vital signs. After removing the tourniquet and blood supply restoration to the limb arises a multifactorial lesion of tissues both ischemic and distant from the site of ischemia. A number of publications have been devoted to the study of morphological disorders in muscle tissue in acute ischemia-reperfusion in the medical literature. However, the researches for effective means for drug correction of these disorders still continues. The aim of the study was to explore peculiarities of skeletal muscle remodeling of the hind limbs of rats, detected by polarization microscopy, in acute ischemia-reperfusion, caused by the application of an arterial tourniquet, and in the correction of reperfusion disorders by carbacetam. Microscopic examination of histological sections of skeletal muscles of the hind limbs of 60 rats below the site of application of the tourniquet under conditions of experimental acute ischemia-reperfusion was performed. Acute ischemia for all animals was caused by application of SWAT rubber bands on the hind limbs of animals, 5–6 mm in width, at the inguinal fold level within 2 hours under thiopental anesthesia. A reperfusion was modeled by removing the tourniquet. Half of the experimental animals in the reperfusion period for the purpose of correction intraperitoneally was administered the nootropic drug 1-oxo-3.3.6-trimethyl-1.2.3.4-tetrahydroindolo[2.3-c]quinoline (carbacetam) at a dose of 5 mg per kilogram of body weight once a day during the entire reperfusion period. The histological specimens of the skeletal muscles were stained with hematoxylin and eosin, and were examined with a light microscope with polarization nozzle. Studies with using the polarization microscopy have shown that in the early reperfusion period morphological criteria for skeletal muscle remodeling expressed by deformation and anisotropy of muscle fibers, disappearance of their transverse striation, cracks and ruptures of fibers, and in the most severe cases there were signs of necrosis of the fibers with their fragmentation into separate lumps. Subject to the correction of reperfusion disorders by carbacetam, there is a decrease in the degree of damage and consistent acceleration of restoration of the skeletal muscles structure, which was the most pronounced in groups of animals with reperfusion terms after 1 and 14 days. Complex of features indicates, that at the tissue level the administration of carbacetam as reduces the ischemic-reperfusion lesion of the muscular fibers, as also accelerates the mechanisms of reparative rhabdomyohistogenesis. Thus, structural changes in the skeletal muscles of the limb after two-hour ischemia and subsequent reperfusion increased in the early reperfusion period and reached its peak after 1 day of reperfusion, and in the late period of reperfusion their reverse development took place. With the correction of disorders by carbacetam, the degree of damage was reduced and the recovery of the skeletal muscle structure of the limb was accelerated.

Comparison of Ischemic Preconditioning and Systemic Piracetam for Prevention of Ischemia-Reperfusion Injury in Musculocutaneous Flaps.

Ischemia-reperfusion injury plays an important role in flap failure. Ischemic preconditioning technique is the only proven method for preventing ischemia-reperfusion injury, but it is not used widely in daily practice because of difficulties such as prolonging the operation time, need for surgical experience, and increasing the risk of complications. This study has been performed with the assumption that piracetam may be a simple and inexpensive alternative to the preconditioning technique due to its antioxidant, antiaggregant, rheological, anti-inflammatory, antiapoptotic, cytoprotective, and immune modulating effects. Thirty-two rats were divided into four groups and latissimus dorsi musculocutaneous flaps were raised. No extra procedure was applied, and no treatment was given to the control group. Four hours of ischemia was created by clamping the thoracodorsal pedicle in the second group. The animals in the third group were treated with 10 minutes of ischemia and reperfusion periods as a preconditioning procedure before the 4 hours of ischemia. Animals in the fourth group received systemic piracetam 30 minutes before and 6 days after reperfusion. Nitric oxide and myeloperoxidase levels in serum and tissue, acute inflammatory cell response, and vascular proliferation in tissue were examined at the postoperative 24th hour and 10th day. Myeloperoxidase activity in both preconditioning and piracetam groups, was significantly lower than the ischemia-reperfusion group. Acute inflammatory cell response was similarly decreased in both preconditioning and piracetam groups compared with ischemia-reperfusion group. Tissue measurements of nitric oxide were also significantly higher in both preconditioning and piracetam groups than in the ischemia-reperfusion group. However, vascular proliferation increased in the preconditioning group, while it did not show any significant change in the piracetam group. This study shows that systemic piracetam treatment provides protection against ischemia-reperfusion injury in musculocutaneous flaps and can offer a simple and inexpensive alternative to the preconditioning technique.

Remote Ischemic Preconditioning Contributes to the Expression of Long Non-Coding RNA H19 and Hypoxia-Inducible Factor-1α during Isolated Coronary Artery Bypass Graft Surgery in Patients with Ischemic Heart Disease

&#x0D; The aim. The aim of our study was to establish the expression levels of long non-coding RNA H19 and hypoxiainducible factor-1α (HIF-1α) in the myocardium and leukocyte fraction as a possible mechanism of adaptation to remote ischemic preconditioning (RIPC) in patients with ischemic heart disease during off-pump isolated coronary artery bypass grafting.&#x0D; Methods. To assess hemodynamic parameters, data from 31 patients (14 in the RIPC group, 17 in control group) were analyzed. The RIPC procedure was performed in patients before surgery by applying a blood pressure cuff to the right forearm. The cuff was inflated to a pressure of 200 mmHg and left for 5 minutes. This was followed by a reperfusion step which lasted 5 minutes. Periods of ischemia and reperfusion lasting 5 minutes were repeated three times. The expression level of long non-coding RNA H19 and HIF-1α was determined in the myocardium and leukocyte fraction by real-time polymerase chain reaction.&#x0D; Results. At the stage of formation of distal anastomoses in patients with RIPC cardiac index (CI) was 24% higher, and stroke volume index (SVI) was 18% higher. Systemic vascular resistance index (SVRI) was significantly lower in patients of the RIPC group (p &lt;0.05). At the end of the operation the CI and SVI levels were significantly higher in the RIPC group, and the rate of SVRI in the RIPC group was significantly lower. In patients with RIPC the expression of H19 in the leukocyte fraction increased by a factor of 3 and in myocardial tissues the level of H19 expression decreased by a factor of 6.7 (p &lt;0.05). The expression of HIF-1α in the myocardium after ischemic preconditioning significantly decreased by a factor of 7.5 (p &lt;0.05), but in the leukocyte fraction there was an insignificant decrease in HIF-1α expression by 20% (p &gt; 0.05).&#x0D; Conclusions. Based on hemodynamic parameters, it can be concluded that patients with RIPC were more hemodynamically stable. Significant changes in the expression of long non-coding RNA H19 and hypoxia-inducible factor-1α demonstrate the importance of these molecules in adaptation to ischemic preconditioning. However, the mechanisms of RIPC involving H19 and HIF-1α need further study.

Effects of endothelin receptor blockade and COX inhibition on intestinal I/R injury in a rat model: Experimental research.

Intestinal ischemia is a highly morbid and mortal condition with no specific treatment. The present study aimed to investigate the effects of cyclooxygenase (COX) inhibition synchronized with nitric oxide (NO) release and endothelin (ET) receptor blockade on oxidative stress, inflammation, vasoconstriction, and bacterial translocation which occur during ischemia-reperfusion (I/R) injury in in-vivo rat intestinal I/R model. 36 male Wistar rats were randomly divided into six groups (n=6). Superior mesenteric artery blood flow (SMABF) was recorded; SMA was occluded for 30min; SMABF was re-recorded at the beginning of the reperfusion phase. Rats were sacrificed after the reperfusion period of 60min. Blood and tissue samples were obtained. Acetylsalicylic acid (ASA), NO-ASA, flurbiprofen (FLUR), and Tezosentan (TS) were administered 15min after ischemia. Histopathological examination, bacterial translocation, and biochemical analysis were performed in plasma and tissue samples. SMABF difference, mean Chiu's score and bacterial translocation were increased in the I/R group and decreased in the treatment groups. Plasma LDH, transaminases, intestinal fatty acid-binding protein (I-FABP), TNF-α, ICAM-1, interferon-gamma (IFN-Ɣ) and proinflammatory cytokine panel; tissue lipid peroxidation, MPO, xanthine oxidase (XO), NO, NF-kB levels and the expression of TNF-α were significantly elevated in the I/R group and markedly decreased in the treatment groups. The tissue antioxidant status was decreased in the I/R group and increased in the treatment groups. It is suggested that NO-ASA, TS, and FLUR can be introduced as promising therapeutics to improve intestinal I/R injury. 2018-29-05 (Animal Experimentations Ethics Committee, Hacettepe University).

Identification of cutaneous perforators for microvascular surgery using hyperspectral technique – A feasibility study on the antero-lateral thigh

Aim of the study was to compare perforator vessel location using color-coded Doppler ultrasound and hyperspectral imaging in the area of the antero-lateral thigh. In a cross-sectional case-control study, the bilateral antero-lateral thigh region was examined for perforator vessel location via color-coded Doppler ultrasound (control) and hyperspectral imaging (test). For hyperspectral imaging, all measurements were conducted without cooling (T0) and after 1 (T1), 2 (T2) and 3 min (T3) of cooling. Additionally, in the reperfusion period after cooling, hyperspectral imaging was conducted at 1, 2 and 3 min (T4/T5/T6). Results from color-coded Doppler ultrasound and hyperspectral imaging were matched at all time points (T0–T6). In total, 71/73 perforator vessel locations could be matched (sensitivity: 97%). Matching of color-coded Doppler ultrasound and hyperspectral imaging was significantly influenced by the cooling protocol and the highest matching values were seen at T3 (3 min cooling; 60 perforator vessels) and T4 (3 min cooling & 1 min reperfusion; 62 perforator vessels) without significant differences (sensitivity 98%; p = 0.9). There were significant differences between T4 and T0, T1 (both p < 0.001), T5 (p = 0.045) and T6 (p = 0.012). For clinical proof of concept, a patient case using a free antero-lateral thigh flap for reconstruction of a facial defect after perforator vessel identification via color-coded Doppler ultrasound and hyperspectral imaging (3 min cooling & 1 min reperfusion) was carried out successfully. In conclusion, hyperspectral imaging potentially offers an additional opportunity for non-invasive, user-independent perforator-site assessment if prior cooling of the site is conducted.

RNA Binding Protein Motif 3 Inhibits Oxygen-Glucose Deprivation/Reoxygenation-Induced Apoptosis Through Promoting Stress Granules Formation in PC12 Cells and Rat Primary Cortical Neurons.

As a sensitive cold-shock protein, RNA binding protein motif 3 (RBM3) exhibits a neuroprotective function in the condition of brain injury. However, how RBM3 is involved in acute ischemic stroke by affecting stress granules (SGs) remains unclear. Here, we established an oxygen-glucose deprivation/reperfusion (OGD/R) model in rat primary cortical neurons and PC12 cells to explore the potential mechanism between RBM3 and SG formation in acute ischemic/reperfusion (I/R) condition. The immunofluorescence results showed that the SG formation significantly decreased in rat primary cortical neurons and PC12 cells during the reperfusion period after 6 h of OGD stimulation. The western blot results, flow cytometry analysis, and cell viability assessment showed that the RBM3 expression and ratio of cell viability significantly decreased, while the rate of apoptosis increased in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Co-immunoprecipitation (Co-IP) and immunofluorescence indicated that RBM3 and GTPase-activating protein-binding protein 1 (G3BP1) colocalized cytoplasm of PC12 cells after 6 h of OGD stimulation when the SGs formation reached the highest level. Besides, overexpression and knockdown of the RBM3 were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. The results of overexpression and knockdown of RBM3 gene illustrated the pivotal role of RBM3 in affecting SG formation and apoptosis level in OGD-treated PC12 cells. In conclusion, RBM3 could combine with G3BP1 resulted in increasing stress granules generation in rat primary cortical neurons and PC12 cells after 6 h of oxygen-glucose deprivation (OGD) injury, which ultimately reduced the apoptosis in OGD-induced cells. Our study may enable a new promising target for alleviating ischemia-reperfusion injury in cells.

Effects of β-adrenergic stimulation on fetal heart rate, heart rate variability, and T-wave elevation during brief umbilical cord occlusions in fetal sheep.

Circulating catecholamines are critical for fetal adaptation to hypoxia by regulating fetal heart rate (FHR) and promoting myocardial contractility and peripheral vasoconstriction. They have been hypothesized to contribute to changes in FHR variability (FHRV) and T-wave morphology, clinical indexes of fetal well-being during labor. β-Adrenergic blockade with propranolol does not affect FHRV during labor-like hypoxemia and only attenuated the increase in T-wave height between the episodes of hypoxemia. To further investigate the potential role of catecholamines, we investigated whether pharmacological β-adrenergic stimulation could increase FHRV and T-wave elevation during intermittent labor-like hypoxemia. Nineteen chronically instrumented fetal sheep at 0.85 of gestation received isoprenaline hydrochloride (n = 7) or saline (control, n = 12), followed by three 1-min complete umbilical cord occlusions (UCOs) separated by 4-min reperfusion periods. Before the UCOs, infusion of isoprenaline increased FHR (P < 0.001), absolute-T/QRS ratio (P < 0.001), and one measure of FHRV [root-mean-square of successive RR interval differences (RMSSD), P < 0.05]. UCOs triggered deep FHR decelerations. During UCOs, isoprenaline was associated with increased FHR (P < 0.001) and absolute-T/QRS ratio (P < 0.05), but no effect on T/QRS ratio was observed when normalized to baseline before UCOs (normalized-T/QRS ratio). Between UCOs, isoprenaline increased FHR (P < 0.001) and absolute-T/QRS ratio (P < 0.05) but did not affect normalized-T/QRS ratio or any measures of FHRV. Arterial pressure was not affected by isoprenaline at any point. Our findings indicate that circulating catecholamines regulate FHR but not FHRV during labor-like hypoxemia and promote T-wave elevation between but not during intermittent fetal hypoxemia.