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Blood clot formation inside the membrane oxygenator (MO) remains a risk in extracorporeal membrane oxygenation (ECMO). It is associated with thromboembolic complications and normally detectable only at an advanced stage. Established clinical monitoring techniques lack predictive capabilities, emphasizing the need for refinement in MO monitoring towards an early warning system. In this study, an MO was modified by integrating four sensor fibers in the middle of the hollow fiber mat bundle, allowing for bioimpedance measurement within the MO. The modified MO was perfused with human blood in an in vitro test circuit until fulminant clot formation. The optical analysis of clot residues on the extracted hollow fibers showed a clot deposition area of 51.88% ± 14.25%. This was detectable via an increased bioimpedance signal with a significant increase 5 min in advance to fulminant clot formation inside the MO, which was monitored by the clinical gold standard (pressure difference across the MO (dp-MO)). This study demonstrates the feasibility of detecting clot growth early and effectively by measuring bioimpedance within an MO using integrated sensor fibers. Thus, bioimpedance may even outperform the clinical gold standard of dp-MO as a monitoring method by providing earlier clot detection. [ABSTRACT FROM AUTHOR]

Background: Previous studies have shown cardiac abnormalities in acute liver injury, suggesting a potential role in the associated high mortality. Methods: We designed an experimental study exploring the short-term effects of acute cholestasis-induced liver injury on cardiac function and structure in a rodent bile duct ligation (BDL) model to elucidate the potential interplay. Thirty-seven male Sprague-Dawley rats were subjected to BDL surgery (n = 28) or served as sham-operated (n = 9) controls. Transthoracic echocardiography, Doppler evaluation of the left anterior descending coronary artery, and myocardial contrast echocardiography were performed at rest and during adenosine and dobutamine stress 5 days after BDL. Immunohistochemical staining of myocardial tissue samples for hypoxia and inflammation as well as serum analysis were performed. Results: BDL animals exhibited acute liver injury with elevated transaminases, bilirubin, and total circulating bile acids (TBA) 5 days after BDL (TBA control: 0.81 ± 2.54 µmol/L vs. BDL: 127.52 ± 57.03 µmol/L; p < 0.001). Concurrently, cardiac function was significantly impaired, characterized by reduced cardiac output (CO) and global longitudinal strain (GLS) in the echocardiography at rest and under pharmacological stress (CO rest control: 120.6 ± 24.3 mL/min vs. BDL 102.5 ± 16.6 mL/min, p = 0.041; GLS rest control: −24.05 ± 3.8% vs. BDL: −18.5 ± 5.1%, p = 0.01). Myocardial perfusion analysis revealed a reduced myocardial blood flow at rest and a decreased coronary flow velocity reserve (CFVR) under dobutamine stress in the BDL animals (CFVR control: 2.1 ± 0.6 vs. BDL: 1.7 ± 0.5 p = 0.047). Immunofluorescence staining indicated myocardial hypoxia and increased neutrophil infiltration. Conclusions: In summary, acute cholestasis-induced liver injury can lead to impaired cardiac function mediated by coronary microvascular dysfunction, suggesting that major adverse cardiac events may contribute to the mortality of acute liver failure. This may be due to endothelial dysfunction and direct bile acid signaling. [ABSTRACT FROM AUTHOR]

The occurrence of thrombus formation within an extracorporeal membrane oxygenator is a common complication during extracorporeal membrane oxygenation therapy and can rapidly result in a life-threatening situation due to arterial thromboembolism, causing stroke, pulmonary embolism, and limb ischemia in the patient. The standard clinical practice is to monitor the pressure at the inlet and outlet of oxygenators, indicating fulminant, obstructive clot formation indicated by an increasing pressure difference (ΔP). However, smaller blood clots at early stages are not detectable. Therefore, there is an unmet need for sensors that can detect blood clots at an early stage to minimize the associated thromboembolic risks for patients. This study aimed to evaluate if forward scattered light (FSL) measurements can be used for early blood clot detection and if it is superior to the current clinical gold standard (pressure measurements). A miniaturized in vitro test circuit, including a custom-made test chamber, was used. Heparinized human whole blood was circulated through the test circuit until clot formation occurred. Four LEDs and four photodiodes were placed along the sidewall of the test chamber in different positions for FSL measurements. The pressure monitor was connected to the inlet and the outlet to detect changes in ΔP across the test chamber. Despite several modifications in the LED positions on the test chamber, the FSL measurements could not reliably detect a blood clot within the in vitro test circuit, although the pressure measurements used as the current clinical gold standard detected fulminant clot formation in 11 independent experiments. [ABSTRACT FROM AUTHOR]

Simple Summary: Animal experiments are essential for the development and optimization of innovative biomedical soft- or hardware. These technologies, not least in the field of intensive care, contribute to an enhanced quality of care and chances of survival. One prominent example that gained public attention due to the COVID-19 pandemic is extracorporeal membrane oxygenation, in short ECMO, as it supports the heart and lungs during severe disease. Even though ECMO is already in use, the components, e.g., the oxygenator that acts as an artificial lung, need further improvement. The basic functions of new devices, e.g., the oxygenation performance, do not necessarily require living animals for the evaluation; therefore, the present study aimed to develop a post-mortem rat cadaver perfusion model exclusively using surplus animals, e.g., from in-house breeding, to reduce the number of animals used for these models, according to the animal welfare principle (the 3Rs: Replace, Reduce, Refine). It was shown that the established rat cadaver model allowed organ perfusion for up to eight hours, making it a promising model for testing new biomedical technology and, thus, a welfare-friendly alternative for existing living rat perfusion models. The development of biomedical soft- or hardware frequently includes testing in animals. However, large efforts have been made to reduce the number of animal experiments, according to the 3Rs principle. Simultaneously, a significant number of surplus animals are euthanized without scientific necessity. The primary aim of this study was to establish a post-mortem rat perfusion model using extracorporeal membrane oxygenation (ECMO) in surplus rat cadavers and generate first post vivo results concerning the oxygenation performance of a recently developed ECMO membrane oxygenator. Four rats were euthanized and connected post-mortem to a venous–arterial ECMO circulation for up to eight hours. Angiographic perfusion proofs, blood gas analyses and blood oxygenation calculations were performed. The mean preparation time for the ECMO system was 791 ± 29 s and sufficient organ perfusion could be maintained for 463 ± 26 min, proofed via angiographic imaging and a mean femoral arterial pressure of 43 ± 17 mmHg. A stable partial oxygen pressure, a 73% rise in arterial oxygen concentration and an exponentially increasing oxygen extraction ratio up to 4.75 times were shown. Considering the 3Rs, the established post-mortal ECMO perfusion rat model using surplus animals represents a promising alternative to models using live animals. Given the preserved organ perfusion, its use could be conceivable for various biomedical device testing. [ABSTRACT FROM AUTHOR]

Clot formation inside a membrane oxygenator (MO) due to blood-to-foreign surface interaction represents a frequent complication during extracorporeal membrane oxygenation. Since current standard monitoring methods of coagulation status inside the MO fail to detect clot formation at an early stage, reliable sensors for early clot detection are in demand to reduce associated complications and adverse events. Bioimpedance analysis offers a monitoring concept by integrating sensor fibers into the MO. Herein, the feasibility of clot detection via bioimpedance analysis is evaluated. A custom-made test chamber with integrated titanium fibers acting as sensors was perfused with heparinized human whole blood in an in vitro test circuit until clot formation occurred. The clot detection capability of bioimpedance analysis was directly compared to the pressure difference across the test chamber (ΔP-TC), analogous to the measurement across MOs (ΔP-MO), the clinical gold standard for clot detection. We found that bioimpedance measurement increased significantly 8 min prior to a significant increase in ΔP-TC, indicating fulminant clot formation. Experiments without clot formation resulted in a lack of increase in bioimpedance or ΔP-TC. This study shows that clot detection via bioimpedance analysis under flow conditions in a blood-perfused test chamber is generally feasible, thus paving the way for further investigation. [ABSTRACT FROM AUTHOR]

Donor organ-shortage has resulted in the increased use of marginal grafts; however, normothermic machine perfusion (NMP) holds the potential for organ viability assessment and restoration of marginal grafts prior to transplantation. Additionally, cell-, oxygen carrier-free and antioxidants-supplemented solutions could potentially prevent adverse effects (transfusion reactions, inflammation, hemolysis), associated with the use of autologous packed red blood cell (pRBC)-based perfusates. This study compared 6 h NMP of porcine kidneys, using an established pRBC-based perfusate (pRBC, n = 7), with the novel cell- and oxygen carrier-free organ preservation solution Ecosol, containing taurine (Ecosol, n = 7). Despite the enhanced tissue edema and tubular injury in the Ecosol group, related to a suboptimal molecular mass of polyethylene glycol as colloid present in the solution, functional parameters (renal blood flow, intrarenal resistance, urinary flow, pH) and oxygenation (arterial pO2, absence of hypoxia-inducible factor 1-alpha) were similar to the pRBC group. Furthermore, taurine significantly improved the antioxidant capacity in the Ecosol group, reflected in decreased lactate dehydrogenase, urine protein and tubular vacuolization compared to pRBC. This study demonstrates the feasibility of 6 h NMP using a taurine containing, cell- and oxygen carrier-free perfusate, achieving a comparable organ quality to pRBC perfused porcine kidneys. [ABSTRACT FROM AUTHOR]

Normothermic machine perfusion (NMP) of kidneys in combination with an optimized perfusate composition may increase donor organ preservation quality, especially in the case of marginal donor grafts. Optimization of currently employed perfusates is still a subject of present research. Due to the advantages of being cell-free, easy to store, and having minimal antigenicity, hemoglobin-based oxygen carriers, such as HBOC-301 (Oxyglobin®, Hemoglobin Oxygen Therapeutics LLC, Souderton, PA, USA), offer an alternative to the commonly used perfusates based on packed red blood cells (pRBC). As previously described, using HBOC results in formation of methemoglobin (metHb) as an adverse effect, inducing hypoxic conditions during the perfusion. As a potential counterpart to metHb formation, the application of the antioxidant ascorbic acid (VitC) is of high interest. Therefore, this study was conducted in four experimental groups, to compare the effect of NMP with (1) HBOC or (3) pRBC, and additionally examine a beneficial effect of VitC in both groups (2) HBOC + VitC and (4) pRBC + VitC. All groups were subjected to NMP for 6 h at a pressure of 75 mmHg. Kidneys in the HBOC groups had a significantly lower renal blood flow and increasing intrarenal resistance, with reduced renal function in comparison to the pRBC groups, as demonstrated by significantly lower creatinine clearance and higher fractional sodium excretion rates. Clinical chemistry markers for tissue damage (LDH, lactate) were higher in the HBOC groups, whereas no significant histological differences were observed. Although the application of VitC decreased oxidative stress levels, it was not able to significantly increase the outcome parameters mentioned above in either group. This study demonstrated that HBOC-301 is inferior to pRBCs in our porcine kidney NMP model, independent of additional VitC administration. Oxidative stress and fragmentation of the hemoglobin polymers could be detected as a possible reason for these results, hence further research, focusing on the use of cell-free oxygen carriers that do not exhibit this complex of issues, is required. [ABSTRACT FROM AUTHOR]

Background: Hemocompatibility of left ventricular assist devices is essential for preventing adverse events. In this study, we compared the hemocompatibility of an axial-flow (Sputnik) to a centrifugal-flow (HeartMate 3) pump. Methods: Both pumps were integrated into identical in vitro test circuits, each filled with 75 mL heparinized human blood of the same donor. During each experiment (n = 7), the pumps were operated with equal flow for six hours. Blood sampling and analysis were performed on a regular schedule. The analytes were indicators of hemolysis, coagulation activation, platelet count and activation, as well as extracellular vesicles. Results: Sputnik induced higher hemolysis compared to the HeartMate 3 after 360 min. Furthermore, platelet activation was higher for Sputnik after 120 min onward. In the HeartMate 3 circuit, the platelet count was reduced within the first hour. Furthermore, Sputnik triggered a more pronounced increase in extracellular vesicles, a potential trigger for adverse events in left ventricular assist device application. Activation of coagulation showed a time-dependent increase, with no differences between both groups. Conclusions: This experimental study confirms the hypothesis that axial-flow pumps may induce stronger hemolysis compared to centrifugal pumps, coming along with larger amounts of circulating extracellular vesicles and a stronger PLT activation. [ABSTRACT FROM AUTHOR]

Increasing evidence indicates that argon has organoprotective properties. So far, the underlying mechanisms remain poorly understood. Therefore, we investigated the effect of argon preconditioning in cardiomyocytes within the first and second window of preconditioning. Primary isolated cardiomyocytes from neonatal rats were subjected to 50% argon for 1 h, and subsequently exposed to a sublethal dosage of hypoxia (<1% O2) for 5 h either within the first (0-3 h) or second window (24-48 h) of preconditioning. Subsequently, the cell viability and proliferation was measured. The argon-induced effects were assessed by evaluation of mRNA and protein expression after preconditioning. Argon preconditioning did not show any cardioprotective effects in the early window of preconditioning, whereas it leads to a significant increase of cell viability 24 h after preconditioning compared to untreated cells (p = 0.015) independent of proliferation. Argon-preconditioning significantly increased the mRNA expression of heat shock protein (HSP) B1 (HSP27) (p = 0.048), superoxide dismutase 2 (SOD2) (p = 0.001), vascular endothelial growth factor (VEGF) (p < 0.001) and inducible nitric oxide synthase (iNOS) (p = 0.001). No difference was found with respect to activation of pro-survival kinases in the early and late window of preconditioning. The findings provide the first evidence of argon-induced effects on the survival of cardiomyocytes during the second window of preconditioning, which may be mediated through the induction of HSP27, SOD2, VEGF and iNOS. [ABSTRACT FROM AUTHOR]

Mortality in critically ill coronavirus disease 2019 (COVID-19) patients is high and pharmacological treatment strategies remain limited. Early-stage predictive biomarkers are needed to identify patients with a high risk of severe clinical courses and to stratify treatment strategies. Macrophage migration inhibitory factor (MIF) was previously described as a potential predictor for the outcome of critically ill patients and for acute respiratory distress syndrome (ARDS), a hallmark of severe COVID-19 disease. This prospective observational study evaluates the predictive potential of MIF for the clinical outcome after severe COVID-19 infection. Plasma MIF concentrations were measured in 36 mechanically ventilated COVID-19 patients over three days after intensive care unit (ICU) admission. Increased compared to decreased MIF was significantly associated with aggravated organ function and a significantly lower 28-day survival (sequential organ failure assessment (SOFA) score; 8.2 ± 4.5 to 14.3 ± 3, p = 0.009 vs. 8.9 ± 1.9 to 12 ± 2, p = 0.296; survival: 56% vs. 93%; p = 0.003). Arterial hypertension was the predominant comorbidity in 85% of patients with increasing MIF concentrations (vs. decreasing MIF: 39%; p = 0.015). Without reaching significance, more patients with decreasing MIF were able to improve their ARDS status (p = 0.142). The identified association between an early MIF response, aggravation of organ function and 28-day survival may open future perspectives for biomarker-based diagnostic approaches for ICU management of COVID-19 patients. [ABSTRACT FROM AUTHOR]

Systemic and localized ischemia and reperfusion injury remain clinically relevant issues after organ transplantation and contribute to organ dysfunctions, among which acute kidney injury is one of the most common. An in vitro test-circuit for normothermic perfusion of porcine kidneys after warm ischemia was used to investigate the antioxidant properties of vitamin C during reperfusion. Vitamin C is known to enhance microcirculation, reduce endothelial permeability, prevent apoptosis, and reduce inflammatory reactions. Based on current evidence about the pleiotropic effects of vitamin C, we hypothesize that the antioxidant properties of vitamin C might provide organ-protection and improve the kidney graft function in this model of ischemia and reperfusion. Methods: 10 porcine kidneys from 5 Landrace pigs were perfused in vitro for 6 h. For each experiment, both kidneys of one animal were perfused simultaneously with a 1:1 mixture of autologous blood and modified Ringer's solution at 38 °C and 75 mmHg continuous perfusion pressure. One kidney was treated with a 500 mg bolus injection of vitamin C into the perfusate, followed by continuous infusion of 60 mg/h vitamin C. In the control test circuit, an equal volume of Ringer's solution was administered as a placebo. Perfusate samples were withdrawn at distinct points in time during 6 h of perfusion for blood gas analyses as well as measurement of serum chemistry, oxidative stress and antioxidant capacity. Hemodynamic parameters and urine excretion were monitored continuously. Histological samples were analyzed to detect tubular- and glomerular-injury. Results: vitamin C administration to the perfusate significantly reduced oxidative stress (49.8 ± 16.2 vs. 118.6 ± 23.1 mV; p = 0.002) after 6 h perfusion, and increased the antioxidant capacity, leading to red blood cell protection and increased hemoglobin concentrations (5.1 ± 0.2 vs. 3.9 ± 0.6 g/dL; p = 0.02) in contrast to placebo treatment. Kidney function was not different between the groups (creatinine clearance vit C: 2.5 ± 2.1 vs. placebo: 0.5 ± 0.2 mL/min/100 g; p = 0.9). Hypernatremia (187.8 ± 4.7 vs. 176.4 ± 5.7 mmol/L; p = 0.03), and a lower, but not significant decreased fractional sodium excretion (7.9 ± 2 vs. 27.7 ± 15.3%; p = 0.2) were observed in the vitamin C group. Histological analysis did not show differences in tubular- and glomerular injury between the groups. Conclusion: Vitamin C treatment increased the antioxidant capacity of in vitro perfused kidney grafts, reduced oxidative stress, preserved red blood cells as oxygen carrier in the perfusate, but did not improve clinically relevant parameters like kidney function or attenuate kidney damage. Nevertheless, due to its antioxidative properties vitamin C might be a beneficial supplement to clinical kidney graft perfusion protocols. [ABSTRACT FROM AUTHOR]

The Pringle maneuver (PM) has been widely used to control blood loss during liver resection. However, hepatic inflow occlusion can also result in hepatic ischemia–reperfusion injury (IRI), especially in patients with a cholestatic, fibrotic, or cirrhotic liver. Here we investigate a nitric oxide synthase (NOS) inhibitor N-Nitroarginine methyl ester (L-NAME) on IRI after the PM and partial hepatectomy of cholestatic livers induced by bile duct ligation (BDL) in rats. Control group (non-BDL/no treatment), BDL + T group (BDL/L-NAME treatment) and BDL group (BDL/no treatment) were analyzed. Cholestasis was induced by BDL in the L-NAME and BDL group and a 50% partial hepatectomy with PM was performed. L-NAME was injected before PM in the BDL + T group. Hepatocellular damage, portal venous flow, microcirculation, endothelial lining, and eNOS, iNOS, interleukin (IL)-6, and transforming growth factor-β (TGF-β) were evaluated. Microcirculation of the liver in the BDL + T group tended to be higher. Liver damage and apoptotic index were significantly lower and Ki-67 labeling index was higher in the BDL + T group while iNOS and TGF-β expression was decreased. This was corroborated by a better preserved endothelial lining. L-NAME attenuated IRI following PM and improved proliferation/regeneration of cholestatic livers. These positive effects were considered as the result of improved hepatic microcirculation, prevention of iNOS formation, and TGF-β mRNA upregulation. [ABSTRACT FROM AUTHOR]

Background: Anesthetic-induced preconditioning (AIP) with volatile anesthetics is a well-known experimental technique to protect tissues from ischemic injury or oxidative stress. Additionally, plasmatic extracellular vesicle (EV) populations and their cargo are known to be affected by AIP in vitro, and to provide organ protective properties via their cargo. We investigated whether AIP would affect the generation of EVs in an in vivo rat model., Methods: Twenty male Sprague Dawley rats received a repetitive treatment with either isoflurane or with sevoflurane for a duration of 4 or 8 weeks. EVs from blood plasma were characterized by nanoparticle tracking analysis, transmission electron microscopy (TEM) and Western blot. A scratch assay (H9C2 cardiomyoblast cell line) was performed to investigate the protective capabilities of the isolated EVs., Results: TEM images as well as Western blot analysis indicated that EVs were successfully isolated. The AIP changed the flotillin and CD63 expression on the EV surface, but not the EV concentration. The scratch assay did not show increased cell migration and/or proliferation after EV treatment., Conclusion: AIP in rats changed the cargo of EVs but had no effect on EV concentration or cell migration/proliferation. Future studies are needed to investigate the cargo on a miRNA level and to investigate the properties of these EVs in additional functional experiments.

The Pringle maneuver (PM) has been widely used to control blood loss during liver resection. However, hepatic inflow occlusion can also result in hepatic ischemia-reperfusion injury (IRI), especially in patients with a cholestatic, fibrotic, or cirrhotic liver. Here we investigate a nitric oxide synthase (NOS) inhibitor N-Nitroarginine methyl ester (L-NAME) on IRI after the PM and partial hepatectomy of cholestatic livers induced by bile duct ligation (BDL) in rats. Control group (non-BDL/no treatment), BDL + T group (BDL/L-NAME treatment) and BDL group (BDL/no treatment) were analyzed. Cholestasis was induced by BDL in the L-NAME and BDL group and a 50% partial hepatectomy with PM was performed. L-NAME was injected before PM in the BDL + T group. Hepatocellular damage, portal venous flow, microcirculation, endothelial lining, and eNOS, iNOS, interleukin (IL)-6, and transforming growth factor-β (TGF-β) were evaluated. Microcirculation of the liver in the BDL + T group tended to be higher. Liver damage and apoptotic index were significantly lower and Ki-67 labeling index was higher in the BDL + T group while iNOS and TGF-β expression was decreased. This was corroborated by a better preserved endothelial lining. L-NAME attenuated IRI following PM and improved proliferation/regeneration of cholestatic livers. These positive effects were considered as the result of improved hepatic microcirculation, prevention of iNOS formation, and TGF-β mRNA upregulation.