Preservation of Mitochondrial Coupling and Renal Function by Controlled Oxygenated Rewarming of Porcine Kidney Grafts

Background: Warm reperfusion after previous cold storage has been shown to have a negative impact on mitochondrial function of organ grafts. Here, we wanted to investigate whether a more controlled warming up of the cold graft by ex vivo machine perfusion with gradually elevated temperature from cold to normothermia (including comparison of two warming up protocols) prior to implantation would be effective in preventing mitochondrial dysfunction upon reperfusion. Methods: All experiments were conducted on porcine kidneys retrieved 15 min after cardiac arrest. After 18 h of cold storage in HTK solution (CS, n = 6), kidneys (n = 6) were subjected to 2 h of reconditioning machine perfusion starting with a hypothermic period followed by a gradual increase in perfusion temperature up to 35 °C (controlled oxygenated rewarming—COR). For a second group (n = 6), the slow warming up was begun instantly after connecting the graft onto the machine (iCOR). Functional recovery of all grafts was then observed upon normothermic reperfusion in vitro. At the conclusion of the experiments, tissue specimens were taken for immediate isolation and analysis of renal mitochondria. Results: COR resulted in a significantly and more than 3-fold increased glomerular filtration rate upon reperfusion, along with a significant higher tubular sodium reabsorption and lesser loss of glucose in comparison to the controls. Enzyme release (AST) was also massively reduced during the reperfusion period. Specific analysis at the mitochondrial level revealed significantly better coupling efficiency and spare respiratory capacity in the COR group compared to the cold storage group. Interestingly, additional experiments revealed that the omission of a hypothermic perfusion period did not deteriorate any of the results after COR, provided that the instant temperature increase from 10 to 35 °C was effectuated in the same controlled manner. Conclusion: Controlled rewarming after extended cold preservation effectively improves mitochondrial recovery upon reperfusion and early functional outcome of kidney grafts.

The diagnostic value of modified systemic ınflammation score in predicting post-operative outcomes of cutaneous melanoma patients who underwent ısolated limb perfusion

Background In-transit metastasis is considered a locoregional disease in cutaneous melanoma (CM) patients. Isolated limb perfusion (ILP) is among the treatment options in selected cases. The aim of this study was to determine the success of pre- and post-perfusion mSIS values in predicting the potential complications and the prognosis of the disease by investigating the early and long-term results of mSIS values calculated before and after ILP in CM cases with in-transit metastases. Materials and methods Patients who underwent ILP within the period from 2014 to 2020 in our department were retrospectively scanned. A total of 20 patients were found to undergo ILP. The scores obtained from modified inflammation score (mSIS) were formulated according to albumin (Alb) and lymphocyte to monocyte ratio (LMR) scores. Results The mean follow-up time was 20.47 months. Complications requiring surgical intervention developed in three patients. According to the Wieberdink local toxicity classification, the majority (70%) of the patients were found to be grade II. Based on pre-perfusion mSIS values, 8 patients were classified as mSIS 0 while six patients were classified as mSIS 1 and 2. Based on post-perfusion mSIS values, 14 patients and one patient were classified as mSIS 2 (70%) and mSIS 0, respectively. Accordingly, univariate analysis showed that mSIS 1 and mSIS 2 were negative prognostic factors for mean survival in the pre-perfusion period (HR 0.162, 95% CI 0.036–0.729; p = 0.018 and HR: 0.223, 95% CI 0.049–1.019; p = 0.053) whereas albumin (Alb) and lymphocyte to monocyte ratio (LMR) were not independent prognostic factors for mean survival. Conclusion The mSIS values calculated in the pre-perfusion period can give an opinion about the OS of the patients whereas post-perfusion mSIS values may predict potential surgical complications and local toxicities.

Increased nitric oxide availability worsens the cardiac performance during early re-perfusion period in adult rats

Objectives Re-perfusion is the standard therapy for acute myocardial infarction, despite the associated pathologies that may contribute to irreversible myocardial injury. The present study aims to clarify the alterations in cardiac activities in response to experimental cardiac ischemic arrest followed by re-perfusion in isolated hearts perfused with nitric oxide (NO) donor, l-arginine, or NO inhibitor, Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), to shed light on the possible role of NO in the re-perfusion process. Methods Hearts isolated from adult Wistar rats were studied on Langendorff preparation under basal conditions and during 30 min re-perfusion following 30 min of total global ischemia. Rats were randomly divided into three groups; control and l-arginine or l-NAME infused heart groups. Cardiac tissue content of malondialdhyde, catalase and nitrite was also measured. Results Compared to the control group, both l-arginine and l-NAME infused hearts showed increased basal chronotropy and myocardial flow rate. Following ischemia and during the whole period of re-perfusion, the three groups demonstrated significant deterioration in the inotropic activity and compromised myocardial flow rate. l-arginine infused hearts revealed depressed inotropy and chronotropy, weak systolic and diastolic functions with compromised myocardial flow at early 5 min of re-perfusion, yet with significantly higher myocardial flow rate by the end of re-perfusion. Conclusions Reducing NO availability by l-NAME revealed mild impact on the ischemia re-perfusion induced contractile dysfunction, whereas excess NO worsens cardiac performance at the early re-perfusion period.

Development of a constant pressure perfused ex vivo model of the equine larynx

Distal axonopathy is seen in a broad range of species including equine patients. In horses, this degenerative disorder of the recurrent laryngeal nerve is described as recurrent laryngeal neuropathy (RLN). The dysfunctional innervation of the cricoarytenoideus dorsalis muscle (CAD) leads to a loss of performance in affected horses. In general, ex vivo models of the larynx are rare and for equine patients, just one short report is available. To allow for testing new therapy approaches in an isolated organ model, we examined equine larynges in a constant pressure perfused setup. In order to check the vitality and functionality of the isolated larynx, the vessels´ reaction to norepinephrine (NE) and sodium nitroprusside (NP) as vasoactive agents was tested. Additionally, the contractility of the CAD was checked via electrical stimulation. To determine the extent of hypoxic alterations, lactate dehydrogenase (LDH) and lactate were measured and an immunofluorescent analysis of hypoxia-inducible factor (HIF-1α), a key transcription factor in hypoxia, was performed. For this, a hypoxia-induced cell culture for HIF-1α was developed. The application of NE led to an expected vasoconstriction while NP caused the expected vasodilation. During a perfusion period of 352 ±20.78 min, LDH values were in the reference range and lactate values slightly exceeded the reference range at the end of the perfusion. HIF-1α nuclear translocation could reliably be detected in the hypoxia-induced cell cultures, but not in sections of the perfused CAD. With the approach presented here, a solid basis for perfusing equine larynges was established and may serve as a tool for further investigations of equine larynx disorders as well as a transferrable model for other species.

NFATc3 is necessary for macrophage iNOS expression during myocardial ischemia and reperfusion

In the present study, we sought to examine whether inhibition NFATc3, improves cardiac function and reduces heart damage during IR. Hearts of male C57BL/J6 and NFATc3 knockout (KO) mice perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion. Our data show that NFATc3 regulates expression of inducible nitric oxide synthase (iNOS) in macrophages stimulated after I/R. In the NFATc3 knockout (KO) mice, left ventricular developed pressure, rate pressure product, contraction and relaxation rates and coronary flow significantly improved following reperfusion compared with C57BL/J6 mice.

Pursuing Ultra-Low VWF Adsorption for Blood-Contacting Devices Under Shear

Many human diseases are treated with blood-contacting medical devices. These devices, which include artificial lung and heart machines and extracorporeal membrane oxygenators (ECMO), save or extend many lives every year. However, the devices are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelet activation and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Attempts have been made to create nonfouling surfaces, and many have in fact have been produced that resist fouling by plasma when tested under non-flow conditions. However, blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated plastic surfaces. VWF normally functions to initiate platelet attachment at sites of vessel injury. Thus, its adsorption to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. We sought to create a surface that achieves ultra-low (< 5 ng/cm2) VWF adsorption under shear, which we anticipate will prevent further protein adsorption and solve the problem of blood and platelet deposition at its initiation. To achieve this objective, we synthesized polymers from the zwitterion sulfobetaine methacrylate (polySBMA) based on published methods. Dopamine was used as the linker between polySBMA and the commonly used materials for devices, resulting in the direct attachment of the polymer to the surface in one step. A more uniform coating of the polymer on certain hydrophobic surfaces was achieved using a mixed solvent of methanol and water. Various coating conditions for polySBMA were tested, including variations in buffer type, salt concentration, polymer concentration, polymer/dopamine ratio, and methanol/water ratio. Eppendorf polypropylene tubes were incubated overnight with polySBMA solution, washed, and then exposed to a VWF solution under shear in a vortexer (3000 rpm, at RT for 90 min). We compared VWF adsorption in the polySBMA-coated PP tubes to adsorption in uncoated tubes. VWF adsorption to tubes coated under optimal conditions was 4% of the adsorption to uncoated tubes, with a VWF surface density below 2 ng/cm2. This coating condition was used for subsequent studies. We next tested the stability of the polySBMA coating after long-term storage. Coated tubes were stored in PBS at 4°C for 1, 4, 7, 28, and 42 days and the integrity of the surface-bound polymers was then examined by evaluating the ability of the stored tubes to resist VWF deposition and comparing their performance to that of freshly coated tubes. Coated tubes stored up to 6 weeks retained their resistance to VWF adsorption under shear (less than 5 ng/cm2), indicating the long-term resilience of the polySBMA coating. To mimic shear stress conditions of blood vessels in capillary, venous, and arterial flow, we used a perfusion system in which a pump (ibidi) generates different flow patterns (continuous unidirectional, oscillating, or pulsatile flow) and shear stresses. Surfaces within the perfusion system were coated with polySBMA to minimize VWF loss. Additional polypropylene test tubing (50 cm long), uncoated or polySBMA-coated, was connected to the perfusion system between two connecters. Purified VWF (2.5 µg/mL in PBS) was perfused through this system for 1 hr. The VWF remaining in the solution was measured by ELISA after the prescribed perfusion period, and multimer composition was compared before and after perfusion. The VWF concentration decreased by 74.1% with uncoated tubing after 1 hr at 20 dyn/cm2, with selective depletion of large VWF multimers. By contrast, in the polySBMA-coated tubing the concentration and multimer composition of VWF was unchanged under identical conditions. In summary, polySBMA effectively prevented VWF adsorption under shear in two types of experiments, and was stable during storage. We expect this coating to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications. Disclosures No relevant conflicts of interest to declare.

NFATc3 is necessary for macrophage iNOS expression during myocardial ischemia and reperfusion

In the present study, we sought to examine whether inhibition NFATc3, improves cardiac function and reduces heart damage during IR. Hearts of male C57BL/J6 and NFATc3 knockout (KO) mice perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion. Our data show that NFATc3 regulates expression of inducible nitric oxide synthase (iNOS) in macrophages stimulated after I/R. In the NFATc3 knockout (KO) mice, left ventricular developed pressure, rate pressure product, contraction and relaxation rates and coronary flow significantly improved following reperfusion compared with C57BL/J6 mice.

Abstract 232: Vascular Dysfunction Induced By High Pressure Levels Is Mediated By Rac-1

Oxidative stress plays a crucial role in the vascular dysfunction present in hypertension. The molecular mechanism involved are not completely elucidated. One of the principal source of oxidative stress at vascular level is the NADPH oxidase enzyme which needs Rac-1 to be activated. Aim of our study was to evaluate the role of Rac-1 in vascular dysfunction induced by high pressure levels. To test this hypothesis, carotid of mice were placed on pressure system and subjected to increasing levels of pressure (100 and 180 mmHg). In particular, after a period of perfusion (1h) with 100 mmHg vessels were subjected to 180 mmHg for three hours. At the end of perfusion period the vascular reactivity was tested. Our results demonstrate that vessels perfused with 180mmHg (n=8) showed an impaired vasorelaxation to acetylcholine as compared to vessels perfused with 100 mmHg (n=7) (% max vasorelaxation: 47±3 vs 88±7, p<0.01). Vasorelaxation to nitroglycerin was comparable between vessels perfused respectively at 100 and 180 mmHg. Some vessels were used to evaluate oxidative stress (n=5) by dihydroethidium and Rac-1 activity (n=8) by Rac-1/PAK complex. Our results demonstrate that high pressure levels induced an increased oxidative stress and more interesting an increased Rac-1 activity as compared to vessels perfused with 100 mmHg. Thus, to clarify the role of Rac-1 in high pressure evoked oxidative stress and vascular dysfunction, some vessels (n=6) were infected with adenoviral vector containing Rac-1 dominant negative. In this experimental setting vessels perfused at 180 mmHg showed an oxidative stress and endothelial vasorelaxation comparable to that observed at 100 mmHg (% max vasorelaxation: 79±6 vs 85±7, n.s.), thus demonstrating a key role of Rac-1 in oxidative stress and endothelial dysfunction evoked by high pressure levels. Thus, Rac-1 could represent the target of novel therapeutic strategies for reducing vascular damage in hypertension.

FVIII production by human lung microvascular endothelial cells

While extrahepatic factor VIII (FVIII) synthesis suffices for hemostasis, the extrahepatic production sites are not well defined. We therefore investigated the ability of the human lungs to produce FVIII. Lungs from heart-beating donors who were declined for transplantation were perfused and ventilated in an isolated reperfusion model for 2 hours. A progressive accumulation of FVIII and von Willebrand factor (VWF) was recorded in the perfusion medium in 3 of 4 experiments. By contrast, factor V, fibrinogen, and immunoglobulin G (IgG) levels remained constant during the perfusion period, indicating that the accumulation of FVIII and VWF was not due to diffusion from the intercellular medium into the vascular system. Purified human lung microvascular endothelial cells produced FVIII during at least 2 passages in vitro. Altogether, these data identify the lung endothelial cells as a FVIII production site in humans.