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INTRODUCTION: Organs procured following brain stem death (BSD) are the main source of organ grafts for transplantation. However, BSD is associated with inflammatory responses that may damage the organ and affect both the quantity and quality of organs available for transplant. Therefore, we aimed to investigate plasma and bronchoalveolar lavage (BAL) pro-inflammatory cytokine profiles and cardiovascular physiology in a clinically relevant 6-hour ovine model of BSD. METHODS: Twelve healthy female sheep (37‒42 Kg) were anaesthetized and mechanically ventilated prior to undergoing BSD induction and then monitored for 6 hours. Plasma and BAL endothelin-1 and cytokines (IL-1β, 6, 8 and tumour necrosis factor alpha (TNF-α)) were assessed by ELISA. Differential white blood cell counts were performed. Cardiac function during BSD was also examined using echocardiography, and cardiac biomarkers (A-type natriuretic peptide and troponin I were measured in plasma. RESULTS: Plasma concentrations big ET-1, IL-6, IL-8, TNF-α and BAL IL-8 were significantly (p < 0.01) increased over baseline at 6 hours post-BSD. Increased numbers of neutrophils were observed in the whole blood (3.1×109 cells/L [95% confidence interval (CI) 2.06 - 4.14] vs. 6×109 cells/L [95%CI 3.92 - 7.97]; p < 0.01) and BAL (4.5×109 cells/L [95%CI 0.41 - 9.41] vs. 26 [95%CI 12.29 - 39.80]; p=0.03) after 6 hours of BSD induction vs baseline. A significant increase in ANP production (20.28 pM [95%CI 16.18 - 24.37] vs. 78.68 pM [95%CI 53.16 - 104.21];p < 0.0001) and cTnI release (0.039 ng/mL vs. 4.26 [95%CI 2.69 - 5.83] ng/mL; p < 0.0001), associated with a significant reduction in heart contractile function, were observed between baseline and 6 hours. CONCLUSIONS: BSD induced systemic pro-inflammatory responses, characterized by increased neutrophil infiltration and cytokine production in the circulation and BAL fluid, and associated with reduced heart contractile function in ovine model of BSD.

Background: Peripheral intravenous catheters (PIVCs) are the most commonly used invasive medical device, yet despite best efforts by end-users, PIVCs experience unacceptably high early failure rates. We aimed to design a new PIVC that reduces the early failure rate of in-dwelling PIVCs and we conducted preliminary tests to assess its efficacy and safety in a porcine model of intravenous access. Methods: We used computer-aided design and simulation to create a PIVC with a ramped tip geometry, which directs the infused fluid away from the vein wall; we called the design the FloRamp™. We created FloRamp prototypes (test device) and tested them against a market-leading device (BD Insyte™; control device) in a highly-controlled setting with five insertion sites per device in four pigs. We measured resistance to infusion and visual infusion phlebitis (VIP) every 6 h and terminated the experiment at 48 h. Veins were harvested for histology and seven pathological markers were assessed. Results: Computer simulations showed that the optimum FloRamp tip reduced maximum endothelial shear stress by 60%, from 12.7 Pa to 5.1 Pa, compared to a typical PIVC tip and improved the infusion dynamics of saline in the blood stream. In the animal study, we found that 2/5 of the control devices were occluded after 24 h, whereas all test devices remained patent and functional. The FloRamp created less resistance to infusion (0.73 ± 0.81 vs 0.47 ± 0.50, p = 0.06) and lower VIP scores (0.60 ± 0.93 vs 0.31 ± 0.70, p = 0.09) than the control device, although neither findings were significantly different. Histopathology revealed that 5/7 of the assessed markers were lower in veins with the FloRamp. Conclusions: Herein we report preliminary assessment of a novel PIVC design, which could be advantageous in clinical settings through decreased device occlusion and reduced early failure rates.

BackgroundPeripheral intravenous catheters (PIVCs) are the most commonly used invasive medical device, yet despite best efforts by end-users, PIVCs experience unacceptably high early failure rates. We aimed to design a new PIVC that reduces the early failure rate of in-dwelling PIVCs and we conducted preliminary tests to assess its efficacy and safety in a large animal model of intravenous access.MethodsWe used computer-aided design and simulation to create a PIVC with a ramped tip geometry, which directs the infused fluid away from the vein wall; we called the design the FloRamp™. We created FloRamp prototypes (test device) and tested them against a market-leading device (BD Insyte™; control device) in a highly-controlled setting with five insertion sites per device in four pigs. We measured resistance to infusion and visual infusion phlebitis (VIP) every six hours and terminated the experiment at 48 hours. Veins were harvested for histology and seven pathological markers were assessed.ResultsComputer simulations showed that the optimum FloRamp tip reduced maximum endothelial shear stress by 60%, from 12.7Pa to 5.1Pa, compared to a typical PIVC tip, and improved the infusion dynamics of saline in the blood stream. In the animal study, we found that 2/5 of the control devices were occluded after 24 hours, whereas all test devices remained patent and functional. The FloRamp created less resistance to infusion (0.73±0.81 vs 0.47±0.50, p=0.06) and lower VIP scores (0.60±0.93 vs 0.31±0.70, p=0.09) that the control device, although neither findings were significantly different. Histopathology revealed that 5/7 of the assessed markers were lower in veins with the FloRamp.ConclusionsAs PIVCs are used in almost every hospitalized patient, there is an urgent need to reduce failure rates. Herein we report preliminary assessment of a novel PIVC design, which could be advantageous in clinical settings through decreased device occlusion.

Use of extracorporeal membrane oxygenation (ECMO) is expanding, however it is still associated with significant morbidity and mortality. Activation of inflammatory and innate immune responses and hemostatic alterations contribute to complications. Hyperoxia may play a role in exacerbating these responses. Nine ex vivo ECMO circuits were tested using fresh healthy human whole blood, with two oxygen levels: 21% inspired fraction of oxygen (FiO ; mild hyperoxia; n=5) and 100% FiO (severe hyperoxia; n=4). Serial blood samples were taken for analysis of platelet aggregometry, leukocyte activation, inflammatory and oxidative stress markers. ECMO resulted in reduced adenosine diphosphate- (p

The acute respiratory distress syndrome (ARDS) describes a heterogenous population of patients with acute severe respiratory failure. However, contemporary advances have begun to identify distinct sub‐phenotypes that exist within its broader envelope. These sub‐phenotypes have varied outcomes and respond differently to several previously studied interventions. A more precise understanding of their pathobiology and an ability to prospectively identify them, may allow for the development of precision therapies in ARDS. Historically, animal models have played a key role in translational research, although few studies have so far assessed either the ability of animal models to replicate these sub‐phenotypes or investigated the presence of sub‐phenotypes within animal models. Here, in three ovine models of ARDS, using combinations of oleic acid and intravenous, or intratracheal lipopolysaccharide, we investigated the presence of sub‐phenotypes which qualitatively resemble those found in clinical cohorts. Principal Component Analysis and partitional clustering identified two clusters, differentiated by markers of shock, inflammation, and lung injury. This study provides a first exploration of ARDS phenotypes in preclinical models and suggests a methodology for investigating this phenomenon in future studies., This study provides a first exploration of ARDS phenotypes in preclinical models and suggests a methodology for investigating this phenomenon in future studies

Right ventricular failure is a common postoperative com-plication following left ventricular assist device (LVAD) im-plantation. Left ventricular assist devices are adapted for right ventricular assist device (RVAD) support by reducing the right pump speed or restricting the diameter of the outflow graft by “banding.” We sought to conduct in vitro hemocompatibility testing in a pulmonary flow condition for current modification of an LVAD for RVAD support, with a specific aim to provide benchmark values for future RVAD development. Two Heart-Ware HVADs coupled to custom-built blood circulation loops, as RVADs, were tested using human blood (n = 6). The RVADs were either used in reduced speed (1,920 ± 50 RPM) or band-ing conditions (3,050 ± 50 RPM) to mimic healthy pulmonary circulation hemodynamics. Blood from the loop was sampled at 0, 15, 60, 150, and 300 min to investigate the level of he-molysis, red blood cell (RBC) deformability, and the activation and aggregation of platelets. The amount of hemolysis and RBC deformability were significantly increased with banding compared with reduced speed (p < 0.05). No significant dif-ferences were found between the two conditions for platelet activation and platelet aggregation. In conclusion, we have evaluated the hemocompatibility of the HVAD when used for RVAD support in the clinically used modes of reduced speed and outflow graft banding under a pulmonary flow conditions that are commonly used in the biventricular failure population. We anticipate the benchmark values in the current study will facilitate future RVAD development.

Background: Extracorporeal membrane oxygenation is a life-saving support for heart and/or lung failure patients. Despite technological advancement, abnormal physiology persists and has been associated with subsequent adverse events. These include thrombosis, bleeding, systemic inflammatory response syndrome and infection. However, the underlying mechanisms are yet to be elucidated. We aimed to investigate whether the different flow dynamics of extracorporeal membrane oxygenation would alter immune responses, specifically the overall inflammatory response, leukocyte numbers and activation/adhesion surface antigen expression. Methods: An ex vivo model was used with human whole blood circulating at 37°C for 6 hours at high (4 L/minute) or low (1.5 L/minute) flow dynamics, with serial blood samples taken for analysis. Results: During high flow, production of interleukin-1β (p Conclusion: We conclude that prolonged circulation at high flow triggers a prominent pro-inflammatory cytokine response and activates neutrophil granule release, but further research is needed to better characterize the effect of flow during extracorporeal membrane oxygenation.

Background Refractory cardiogenic shock (CS), frequently complicated by pulmonary failure, often requires veno-arterial extracorporeal membrane oxygenation (VA-ECMO) to sustain end-organ perfusion. Currently available animal models, such as the coronary ligation model, result in highly variable injury profiles and unacceptably high levels of subsequent ventricular fibrillation, cardiac arrest, and death. As the use of ECMO increases, there is a growing need for a clinically relevant, robust, and titratable model of severe cardiopulmonary failure supported by VA-ECMO. Methods Six sheep (60 ± 6 kg) were anaesthetized, intubated and mechanically ventilated. VA-ECMO was initially carried out at a flow rate of 1 L/min. CS was induced through 1-mL left ventricle myocardial injections of 96% ethanol and confirmed when systolic blood pressure (SBP) was 4 mmol/L. Then, pulmonary failure was confirmed when PaO2 was

The Acute Respiratory Distress Syndrome (ARDS) describes a heterogenous population of patients with acute severe respiratory failure. However, contemporary advances have begun to identify distinct sub-phenotypes that exist within its broader envelope. These sub-phenotypes have varied outcomes and respond differently to several previously studied interventions. A more precise understanding of their pathobiology and an ability to prospectively identify them, may allow for the development of precision therapies in ARDS. Historically, animal models have played a key role in translational research, although few studies have so far assessed either the ability of animal models to replicate these sub-phenotypes or investigated the presence of sub-phenotypes within animal models. Here, in three ovine models of ARDS, using combinations of oleic acid and intravenous, or intratracheal lipopolysaccharide, we demonstrate the presence of sub-phenotypes which qualitatively resemble those found in clinical cohorts. Principal Components Analysis and partitional clustering reveal two clusters, differentiated by markers of shock, inflammation, and lung injury. This study provides the first preliminary evidence of ARDS phenotypes in pre-clinical models and develops a methodology for investigating this phenomenon in future studies.

Background A lung transplant is the last resort treatment for many patients with advanced lung disease. The majority of donated lungs come from donors following brain death (BD). The endothelin axis is upregulated in the blood and lung of the donor after BD resulting in systemic inflammation, lung damage and poor lung graft outcomes in the recipient. Tezosentan (endothelin receptor blocker) improves the pulmonary haemodynamic profile; however, it induces adverse effects on other organs at high doses. Application of ex vivo lung perfusion (EVLP) allows the development of organ-specific hormone resuscitation, to maximise and optimise the donor pool. Therefore, we investigate whether the combination of EVLP and tezosentan administration could improve the quality of donor lungs in a clinically relevant 6-h ovine model of brain stem death (BSD). Methods After 6 h of BSD, lungs obtained from 12 sheep were divided into two groups, control and tezosentan-treated group, and cannulated for EVLP. The lungs were monitored for 6 h and lung perfusate and tissue samples were processed and analysed. Blood gas variables were measured in perfusate samples as well as total proteins and pro-inflammatory biomarkers, IL-6 and IL-8. Lung tissues were collected at the end of EVLP experiments for histology analysis and wet-dry weight ratio (a measure of oedema). Results Our results showed a significant improvement in gas exchange [elevated partial pressure of oxygen (P = 0.02) and reduced partial pressure of carbon dioxide (P = 0.03)] in tezosentan-treated lungs compared to controls. However, the lungs hematoxylin–eosin staining histology results showed minimum lung injuries and there was no difference between both control and tezosentan-treated lungs. Similarly, IL-6 and IL-8 levels in lung perfusate showed no difference between control and tezosentan-treated lungs throughout the EVLP. Histological and tissue analysis showed a non-significant reduction in wet/dry weight ratio in tezosentan-treated lung tissues (P = 0.09) when compared to control. Conclusions These data indicate that administration of tezosentan could improve pulmonary gas exchange during EVLP.

Background Heart failure is an inexorably progressive disease with a high mortality, for which heart transplantation (HTx) remains the gold standard treatment. Currently, donor hearts are primarily derived from patients following brain stem death (BSD). BSD causes activation of the sympathetic nervous system, increases endothelin levels, and triggers significant inflammation that together with potential myocardial injury associated with the transplant procedure, may affect contractility of the donor heart. We examined peri-transplant myocardial catecholamine sensitivity and cardiac contractility post-BSD and transplantation in a clinically relevant ovine model. Methods Donor sheep underwent BSD (BSD, n = 5) or sham (no BSD) procedures (SHAM, n = 4) and were monitored for 24 h prior to heart procurement. Orthotopic HTx was performed on a separate group of donor animals following 24 h of BSD (BSD-Tx, n = 6) or SHAM injury (SH-Tx, n = 5). The healthy recipient heart was used as a control (HC, n = 11). A cumulative concentration-effect curve to (-)-noradrenaline (NA) was established using left (LV) and right ventricular (RV) trabeculae to determine β1-adrenoceptor mediated potency (-logEC50 [(-)-noradrenaline] M) and maximal contractility (Emax). Results Our data showed reduced basal and maximal (-)-noradrenaline induced contractility of the RV (but not LV) following BSD as well as HTx, regardless of whether the donor heart was exposed to BSD or SHAM. The potency of (-)-noradrenaline was lower in left and right ventricles for BSD-Tx and SH-Tx compared to HC. Conclusion These studies show that the combination of BSD and transplantation are likely to impair contractility of the donor heart, particularly for the RV. For the donor heart, this contractile dysfunction appears to be independent of changes to β1-adrenoceptor sensitivity. However, altered β1-adrenoceptor signalling is likely to be involved in post-HTx contractile dysfunction.

Background Extracorporeal membrane oxygenation (ECMO) is a life-saving modality used to manage cardiopulmonary failure refractory to conventional medical and surgical therapies. Despite advances in ECMO equipment, bleeding and thrombosis remain significant complications. While the flow rate for ECMO support is well recognized, less is known about the minimum-rate requirements and haemostasis. We investigated the relationship between different ECMO flow rates, and their effect on haemolysis and coagulation. Methods Ten ex-vivo ECMO circuits were tested using donated

Purpose Cold static storage (CSS) is the standard method for heart preservation during transplantation (HTx). However, CSS beyond 4 hours increases the risk of primary graft dysfunction (PGD). Hypothermic ex vivo perfusion (HEVP) of donor hearts allows oxygen delivery during preservation, and may facilitate extended donor preservation without increasing PGD risk. We compared post-HTx survival, systemic inflammation and cardiac function following donor heart preservation by CSS (2 hrs) versus HEVP (2 and 8 hrs). Methods Brain death was induced in donor sheep for 24 hrs. Donor hearts were preserved by a) CSS for 2 hrs (n=7), b) HEVP for 2 hrs (n=4), or c) HEVP for 8 hrs (n=4). Orthotopic HTx was performed in matched recipients. Recipients were weaned from cardiopulmonary bypass and monitored for 6 hrs. Recipient blood was collected and assayed for inflammatory cytokines and cardiac markers. Cardiac function was assessed by echocardiography. Results Six-hour survival was 71% following CSS, and 100% following 2 and 8 hrs HEVP, respectively. Recipients systemic interleukin-6 and 8 levels were reduced using HEVP vs CSS. Post-HTx haemodynamic function was no different between groups, but HEVP reduced the requirement for vasoactive support compared to CSS (2 hrs CSS: 1.57±0.7; 2 hrs HEVP: 0.35±0.09; 8 hrs HEVP: 0.35±0.05 mmHg−1). HEVP was associated with reduced post-HTx lactate (2 hrs CSS: 11.4±1.8; 2 hrs HEVP: 5.2±0.7; 8 hrs HEVP: 6.7±1.3 mmol/L), more stable base excess and physiological pH in blood. Post-HTx cardiac function was no different between groups. Cardiac troponin I levels were comparable between CSS vs. 8 hrs HEVP, but reduced with 2 hrs HEVP. Conclusion Preliminary data on donor heart preservation by HEVP shows promising outcomes in comparison to CSS. Heart preservation by HEVP can be extended up to 8 hours, without compromising post-HTx recipient survival. HEVP may assist in overcoming limitations in preservation time associated with HTx, without increasing PGD risk.

There is growing evidence that inflammation underpins many common diseases. Inflammatory/immunomodulatory/immune mediators, such as cytokines, are key modulators of inflammation and mediate both immune cell recruitment and complex intracellular signalling pathways. Ovine models of disease are increasingly utilized in pre-clinical research, however existing methods for measuring cytokine levels are limited. We established and validated enzyme-linked immunosorbent assays (ELISAs) targeting interleukin (IL)-1β, IL-6, IL-8 and IL-10 in sheep plasma. These ELISAs showed high sensitivity and specificity with intra- and inter-assay CV's below 10%, and recovery rates between 82 and 123%. Sensitivity for IL-1β, IL-6, IL-8 and IL-10 were 117.6 pg/mL, 443.1 pg/mL, 30.9 pg/mL, and 64.3 pg/mL, respectively. ELISA test result reproducibility decreased significantly after 12 weeks of plasma storage at -80 °C. Therefore, for accurate cytokine measurements, plasma samples need to be tested within three months of sample collection to account for cytokine protein degradation. These ELISAs offer a reliable and convenient method to identify inflammatory cytokine changes in sheep, allowing key insights into the disease pathogenesis of these ruminants.

Purpose Brain death (BD) causes metabolic and energetic imbalances leading to cardiac dysfunction, and predisposes the donor heart to further injury following heart transplantation (HTx). The metabolic mechanisms required for myocardial energy production during BD and subsequent HTx are poorly understood. Our aim was to determine the myocardial metabolic profile and mitochondrial function following donor BD and HTx. Methods Donor BD in sheep was induced by inflation of a catheter placed through the skull (catheter placement, but no inflation for SHAM), followed by 24 hrs monitoring, and heart procurement (n=6/group, BD vs. SHAM). Additional donor hearts exposed to BD/SHAM were flushed with cold St Thomas cardioplegia, and stored via cold static storage (CSS) for ∼2 hrs. Following standard orthotopic HTx, recipients were weaned off bypass and monitored for ≤6 hrs prior to heart procurement (n=4/group, BD-Tx vs. Sh-Tx). Cardiac mitochondrial function was assessed using high resolution respirometry. Metabolic profiles were determined in hearts using metabolomics. Cardiac mitochondrial function was also determined in two sheep that underwent HTx following BD and 8 hr hypothermic ex vivo perfusion (HEVP) preservation. Results BD caused significant right ventricular (RV) mitochondrial uncoupling (vs. SHAM). HTx following CSS also impaired RV mitochondrial function, with these effects more pronounced in hearts exposed to both donor BD and HTx. Early findings show that HEVP improved cardiac mitochondrial function post-HTx (vs. CSS). Metabolically, BD increased myocardial amino-acid utilisation and accumulation of glucose metabolites. Post-HTx, particularly in those exposed to donor BD, there was a significant decrease in metabolites involved in mitochondrial respiration (eg. NAD, Acetyl-CoA) and accumulation of fatty acids and xanthine (purine breakdown). Conclusion BD appears to trigger cardiac mitochondrial uncoupling. This may be a protective mechanism against higher amino-acid utilisation and glucose accumulation, in order to maintain adequate mitochondrial function for cell survival. HTx following CSS, particularly from BD donors, induces significant mitochondrial dysfunction, which occurs in response to upstream metabolic impairments. Strategies that improve cardiac mitochondrial function or metabolism (eg. HEVP) may assist to improve HTx outcomes.

Purpose Brain stem death (BD) causes increased levels of catecholamines which may induce β-adrenoceptor desensitisation and mitochondrial dysfunction. Combined, this contributes to donor heart dysfunction (DHD) and post-transplant graft failure. We sought to examine peri-transplant catecholamine sensitivity, cardiac contractility and mitochondrial function post-BD and transplantation (Tx) in a clinically relevant ovine model. Methods Donor sheep underwent BD (BD n = 6) or sham instrumentation (SH, n = 4) and monitored for 24 h followed by heart procurement and cold static storage (CSS). Orthotopic heart transplantation (HTx) was performed after CSS with BD (BD-Tx, n = 7) or SH donors (SH-Tx, n = 4). Comparisons were made with the excised healthy recipient heart (HR, n = 9). A cumulative concentration-effect curve to (−)-noradrenaline (NA) was established using left and right ventricular trabeculae to determine β1-adrenoceptor mediated potency and contractility. Phenoxybenzamine (5 μM) and ICI118551 (50 nM) were used to block α- and β2-adrenoceptors, respectively. Mitochondrial respirometry was assessed using the Oroboros Oxygraph in the presence of carbohydrate (CHO) or fatty acid (FAO) substrates. Results BD caused a significant decrease in RV (but not LV) contractility with preserved sensitivity to NA, but an increase in mitochondrial proton slip (in LEAK state). Both ventricles showed a significant decrease in complex-II respiration for CHO and FAO substrates. HTx (SH-Tx and BD-Tx) showed significantly reduced RV contractility and a slight decrease in NA sensitivity. This was in conjunction with mitochondrial dyscoupling evidenced by significantly higher RV proton slip, and depressed LV and RV complex-II respiration for both substrates. Conclusion We have shown that β1-adrenoceptor desensitisation doesn't occur as a result of BD but observed post-Tx, in BD and SH donors. Mitochondrial dysfunction however, plays a significant role in DHD and post-transplant graft failure. These results have significant implications on patient management, highlighting mitochondria as an important contributor to cardiac dysfunction and as a therapeutic target.

Introduction: Mitochondria are essential for adequate cardiovascular function and are important in the generation of reactive oxygen species (ROS). However, mitochondrial function and ROS post donor brain stem death (BD) and heart transplantation HTx is poorly understood and may contribute to donor heart dysfunction (DHD) and post HTx graft failure (GF). Objectives∖Aims: To examine mitochondrial function and ROS production in hearts both after 24hrs of BSD and post HTx. Methods: Donor sheep underwent BD (BD n=13) or sham instrumentation (SH, n=8) and monitored for 24hrs followed by heart procurement or cold static storage. Orthotopic HTx followed, in 7∖13 BD (BD-Tx) and 4/8 SH donors (SH-Tx). Mitochondrial respirometry was assessed using the Oroboros Oxygraph using carbohydrate (CHO) or fatty acid (FAO) substrates for both ventricles. O2 flux control ratios (FCR) were calculated in LEAK (no ADP) and Complex- I (CI) & II (CII) OXPHOS (with ADP) respiration states. Tissue levels of 3-nitrotyrosine and glutathione:reduced glutathione (GSH:GSSG) ratio were assessed using plate based assays. Results: BD caused a significant increase in LEAK RV proton slip. Both ventricles showed a trend toward higher O2 consumption at CI (known driver of ROS) and a significant decrease in CII respiration for both substrates. Post HTx showed mitochondrial dyscoupling evidenced by a trend toward higher CI O2 consumption, significantly higher RV proton slip, and depressed LV and RV CII respiration. For the LV, GSH:GSSG ratios were significantly lower for BD, BD-Tx and SH-Tx. For the RV, GSH:GSSG ratios were non-significantly depressed post BD both pre and post Tx. Conclusions: We have shown that donor BD and Tx caused significant mitochondrial dyscoupling and higher oxidative stress. Mitochondrial function, particularly CI ROS production may then contribute to DHD and consequently post-HTx GF. Therapeutically targeting mitochondrial ROS could improve patient prognosis.