Normothermic Ischemia Research Articles

Mild hypothermia attenuates ischaemia/reperfusion injury: insights from serial non-invasive pressure-volume loops.

Mild hypothermia, 32-35°C, reduces infarct size in experimental studies, potentially mediating reperfusion injuries, but human trials have been ambiguous. To elucidate the cardioprotective mechanisms of mild hypothermia, we analysed cardiac performance in a porcine model of ischaemia/reperfusion, with serial cardiovascular magnetic resonance (CMR) imaging throughout 1 week using non-invasive pressure-volume (PV) loops. Normothermia and Hypothermia group sessions (n = 7 + 7 pigs, non-random allocation) were imaged with Cardiovascular magnetic resonance (CMR) at baseline and subjected to 40 min of normothermic ischaemia by catheter intervention. Thereafter, the Hypothermia group was rapidly cooled (mean 34.5°C) for 5 min before reperfusion. Additional CMR sessions at 2 h, 24 h, and 7 days acquired ventricular volumes and ischaemic injuries (unblinded analysis). Stroke volume (SV: -24%; P = 0.029; Friedmans test) and ejection fraction (EF: -20%; P = 0.068) were notably reduced at 24 h in the Normothermia group compared with baseline. In contrast, the decreases were ameliorated in the Hypothermia group (SV: -6%; P = 0.77; EF: -6%; P = 0.13). Mean arterial pressure remained stable in Normothermic animals (-3%, P = 0.77) but dropped 2 h post-reperfusion in hypothermic animals (-18%, P = 0.007). Both groups experienced a decrease and partial recovery pattern for PV loop-derived variables over 1 week, but the adverse effects tended to attenuate in the Hypothermia group. Infarct sizes were 10 ± 8% in Hypothermic and 15 ± 8% in Normothermic animals (P = 0.32). Analysis of covariance at 24 h indicated that hypothermia has cardioprotective properties incremental to reducing infarct size, such as higher external power (P = 0.061) and lower arterial elastance (P = 0.015). Using non-invasive PV loops by CMR, we observed that mild hypothermia at reperfusion alleviates the heart's work after ischaemia/reperfusion injuries during the first week and preserves short-term cardiac performance. This hypothesis-generating study suggests hypothermia to have cardioprotective properties, incremental to reducing infarct size. The primary cardioprotective mechanism was likely an afterload reduction acutely unloading the left ventricle.

Hypothermia yields favourable cardiac energetics in experimental ischemia/reperfusion – insights from non-invasive pressure-volume loops by CMR

Abstract Background/Introduction In experimental studies, mild hypothermia (<35°C) has reduced infarct size [1], potentially through reduced reperfusion injury [2], but human trials have been ambiguous [3]. Furthermore, a pressure-volume (PV) loop framework is the gold standard for assessing cardiac performance, but the invasive approach limits its clinical use. Therefore, we used a porcine model of ischemia/reperfusion with cardiovascular magnetic resonance (CMR) imaging throughout one week to acquire non-invasive PV loops. Purpose To quantify the cardioprotective effects of hypothermia at reperfusion by serial CMR imaging in a perspective of cardiac energy and mechanics. Methods Normothermia and Hypothermia groups (n=7+7 pigs) were imaged with CMR at baseline and subjected to 40 minutes of normothermic ischemia by catheter intervention. Thereafter, the Hypothermia group was rapidly cooled (<35°C) for 5 minutes before reperfusion. Additional imaging at two hours, 24 hours, and seven days were used to assess ventricular volumes and ischemic injuries. Infarct size was determined by high-resolution ex-vivo T1-weighted images. Results Figure 1 shows that stroke volume (SV: p=0.029; Friedmans test) and ejection fraction (EF: p=0.068) were notably reduced post-reperfusion in the Normothermia group. In contrast, the decreases were ameliorated in the Hypothermia group (SV: p=0.77; EF: p=0.13). Mean arterial pressure (MAP) was stable in the Normothermia group (p=0.77) but dropped two hours post-reperfusion in hypothermic animals (p=0.007). Over one week, both groups experienced the same decrease and partial recovery pattern for stroke work, external power, and ventricular-arterial coupling. Still, the adverse effects from ischemia were generally attenuated in the Hypothermia group. Although not significant, ischemia/reperfusion caused smaller infarcts in the Hypothermia group (10±8% vs 15±8%; p=0.32). The change in PV loop variables from baseline to 24 hours was compared with infarct size for each group. Though statistical power was limited, the general trend in analyses of covariance indicated that hypothermia has cardioprotective properties incremental to reducing infarct sizes (Figure 2), such as higher external power (p=0.061) and lower arterial elastance (p=0.015). Conclusion Using non-invasive PV loops by CMR, we observed that mild hypothermia before reperfusion results in favourable cardiac energetics that alleviates the heart's work after an ischemia/reperfusion injury during the first week. This study suggests that hypothermia has cardioprotective properties, incremental to reducing infarct size. The primary cardioprotective mechanism was likely an afterload reduction unloading the injured left ventricle. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): The Swedish Heart-Lung FoundationThe Medical Faculty of Lund University (ALF)

Tetrandrine Protects Hearts Against Ischemia‐Reperfusion Injury Via The Inhibition of p66Shc Phosphorylation And Mitochondrial Translocation

Introduction Increased Mitochondrial (Mito) reactive oxygen species (ROS) generation is a major cause of cardiac ischemia/reperfusion (I/R) injury. p66 Shc is an adaptor protein that regulates multiple pathways governing Mito redox state and ROS generation. Tetrandrine (TET), a bisbenzylisoquinoline alkaloid that is extracted from the root of Stephania tetrandra S. Moore, has been used to treat cardiovascular diseases and hypertension. Here we investigate whether Tet exerts cardioprotection in I/R injury and its underlying mechanism mechanisms. Here we studied the effects of TET on phosphorylation and Mito translocation of p66 Shc, Mito oxidative stress/damage, cardiac function and infarct size in mouse heart model of I/R injury. Methods: Hearts (n = 14 each) isolated from C57BL/6 mice were perfused in the Langendorf mode. After 30-min equilibration, the heart was treated with TET or vehicle for 10 min, then subjected to global, no-flow normothermic ischemia for 30 min followed by reperfusion with the corresponding treatment for 120 min. Cardiac function and myocardial energy metabolism were measured with 31 P-NMR spectroscopy continuously and simultaneously. Effluent was collected to detect the release of lactate dehydrogenase. At the end of the reperfusion, half of the hearts were stained with 2,3,5-triphenyltetrazolium chloride to evaluate infarct size, whereas the other half were freeze-clamped and whole protein lysate, cytosolic and Mito fractions were prepared from the clamped tissues to assess the phosphorylation at ser36 and Mito translocation of p66 SHC. Mito H2O2, malondialdehyde (MDA), and protein carbonyls were measured to assess ROS generation and oxidative damage to lipids and proteins, respectively. Results 3 μM TET significantly reduced the heart infarct size and improved cardiac functional recovery after I/R injury. Consistently, TET reversed I/R-induced increases in H2O2, MDA, and protein carbonyls. Interestingly, 3 μM TET significantly decreased the I/R-induced phosphorylation of p66 Shc at serine 36 and its translocation to mitochondria. Conclusions: Our data indicate that TET protects the heart against myocardial I/R injury by preventing Mito oxidative damage via mechanisms that are likely to suppressing p66 Shc phosphorylation and Mito accumulation. Targeting p66 Shc phosphorylation and translocation may serve as new therapeutic strategies for I/R injury.

Evaluation of the suitability of a donor heart for transplantation after various asystole periods in experiment

The lack of donor hearts for transplantation in patients with end-stage chronic heart failure makes us look for new ways to increase the pool of donors. One of the possible ways to increase the donor base may be the use of asystolic donors, i. e. patients with in-hospital cardiac arrest or patients with cardiac arrest after brain death. The aim of the study was to examine the morphofunctional state of the heart after various periods of normothermic asystole in an experiment on rats. Materials and methods . Experiments were performed on female Wistar rats. After cervical dislocation and registration of respiratory arrest, the body temperature of the rat was maintained above at 36.5±0.5 °C for 10, 15 and 20 minutes, depending on the group. After asystole period, the heart was connected to the Langendorff apparatus to provide perfusion for 90 minutes. During perfusion of the heart, the pulse pressure inside the left ventricle (LVPP) and the volumetric flow rate of coronary perfusion (CP) were recorded every 15 minutes. After the end of the experiment, the volume of necrosis was calculated. Results . In the group with 20-minute ischemia, there was no recovery of ventricular contractions, the volume of necrosis was 34,9±11,7 %. In the group with 15-minute ischemia, the volume of necrosis was 19,5±7,6 %. In the group with 10-minute normothermic ischemia, the volume of necrosis was 13,1±5,1 %. Conclusions . 15 minutes of normothermic cardiac arrest in situ causes irreversible damage to the heart of the animal, which does not allow us to consider it as a potential donor heart. However, this volume of damage to the myocardium and microvasculature, together with the restoration of the contractile activity of the heart, makes this group the main one for the continuation of such experiments using various methods of heart reconditioning.

Endothelial cell-derived extracellular vesicles exert cardio-protective effect via their protein cargo

Abstract Background Extracellular vesicles (EV) are recognized as carriers of relevant biological effects and have been identified as regulators of cell-to-cell communication contributing to several patho-physiological processes. These processes include angiogenesis/coagulation/tissue repair/inflammation. In ischemia/reperfusion (I/R) settings, along with the direct effects of the I/R itself, paracrine mechanisms associated with the activation of the inflammatory response, primary involving endothelial cells, are crucial drivers of both vessel and cardiomyocyte damage. Purpose Since in models of myocardial I/R injury the role of EV released from endothelial cells is still unclear, our hypothesis was to provide insight on this specific topic. To this end, naïve endothelial cell (EC)-derived EV (eEV) and eEV released in response to the pro-inflammatory cytokine interleukin-3 (IL-3) (eEV-IL-3) have been evaluated on different I/R models. Methods eEV were characterized by MACSPlex-Exosome-Kit and western blot analysis. For the in-vitro hypoxia-reoxygenation (H/R) experiments, H9c2 or EC were pretreated with eEV, eEV-IL-3 (1x104 EV/cell) or IL-3 (10ng/ml) for 2 hours and then exposed to hypoxia (1% O2, 5% CO2) for additional 2 hours in the presence of eEV, eEV-IL-3 or IL-3 and subsequently reoxygenated (21% O2 and 5% CO2) for 1 hour. To verify the effect of EC treated with eEV, eEV-IL-3 or IL-3 on H9c2 and subjected to H/R protocol, transwell assay was used. At the end of the H/R protocol, cell viability was assessed. For ex-vivo experiments, isolated rat hearts, pretreated with a buffer containing EV (from EC pretreated or not with IL-3), were subjected to 30 minutes global normothermic ischemia and 1 hour reperfusion. Triton infusion was also used as a model of endothelial damage. At the end of I/R, the infarct size was measured and expressed as a percentage of total left ventricular mass (LVM). The role of eNOS/guanylyl-cyclase/MEK1/2 pathways in mediating eEV biological effects was also evaluated using different inhibitors both in in-vitro and ex-vivo models. Finally, protein profiles of eEV and eEV-IL-3 were analyzed using label free mass spectrometry. Results eEV and eEV-IL-3 protect EC, but not H9c2 exposed to H/R protocol, while eEV, but not eEV-IL-3-treatment limits I/R injury in the rat heart. Rat hearts pre-treated with triton significantly avoid eEV-induced cardio-protection. Transwell assay showed a reduction of H9C2 mortality after treatment with both eEV and eEV-IL-3. Proteomic analysis revealed that MEK1/2 and the endothelial-NOS (eNOS)-antagonist caveolin-1 were differentially expressed in eEV and eEV-IL-3. The use of eNOS/guanylyl-cyclase/MEK1/2 inhibitors prevented eEV-induced cardio-protection. Conclusions These observations indicate that eEV, but not eEV-IL-3, have cardio-protective effects when given as preconditioning agents. We have also shown that the activation of eNOS/GC/MEK1/2 pathway is crucial for eEV-mediated cardio-protection. Funding Acknowledgement Type of funding source: None

Mechanical Reperfusion Following Prolonged Global Cerebral Ischemia Attenuates Brain Injury

Previous experiments demonstrated improved outcome following prolonged cerebral ischemia given controlled brain reperfusion using extracorporeal circulation. The current study further investigates this. Young adult pigs were exposed to 30 min of global normothermic cerebral ischemia, achieved through intrathoracic clamping of cerebral arteries, followed by 20 min of isolated mechanical brain reperfusion. Leukocyte-filtered blood was delivered by a roller-pump at fixed pressure and flow. One experimental group additionally had a custom-made buffer solution delivered at 1:8 ratio with the blood. Hemodynamics including intracranial pressure were monitored. Blood gases were from peripheral arteries and the sagittal sinus, and intraparenchymal brain microdialysis was performed. The brains were examined by a neuropathologist. The group with the added buffer showed lower intracranial pressure as well as decreased intraparenchymal glycerol and less signs of excitotoxicity and ischemia, although histology revealed similar degrees of injury. A customized mechanical reperfusion improves multiple parameters after prolonged normothermic global cerebral ischemia.Graphical The current study investigates if it possible to improve neurological outcomes following prolonged global brain ischemia. The results indicate that a customized mechanical reperfusion protocol can attenuate neurological injury.

Hypothermia Inhibits the Expression of Receptor Interacting Protein Kinases 1 and 3 After Transient Spinal Cord Ischaemia in Rabbits

Necroptosis, a form of regulated necrosis, might be a potential mechanism of delayed paraplegia; therefore, its role in transient spinal cord ischaemia was investigated by immunohistochemical analysis of necroptosis related protein receptor interacting protein kinase (RIP) 1, RIP3, and cellular inhibitor of apoptosis protein (cIAP) 1/2. This study used rabbit normothermic (n=24) and hypothermic (n=24) transient spinal cord ischaemia models and sham controls (n=6). Neurological function was assessed according to a modified Tarlov score at 8h, 1, 2, and 7 days after reperfusion (n=6 each). Morphological changes in the spinal cord were examined using haematoxylin and eosin staining in the sham, 2, and 7 day groups. Western blot and histochemical analyses of RIP1, RIP3, and cIAP1/2, and double label fluorescent immunocytochemical studies of RIP3 and cIAP1/2 were performed at 8h, 1, and 2 days after reperfusion (n=6 each). There were significant differences in neurological function between the normothermic and hypothermic groups (median scores 0 and 5at 7 days, p=.023). In the normothermic group, most motor neurons were lost seven days after reperfusion (p=.046 compared with sham), but they were preserved in the hypothermic group. Western blot analysis revealed the upregulation of RIP1, RIP3, and cIAP1/2at 8h in the normothermic group (RIP1, p=.032; RIP3, p<.001; cIAP1/2, p=.041 compared with sham), and the overexpression of RIP3 was prolonged for two days. In the hypothermic group, the expression of these proteins was not observed. The double label fluorescent immunocytochemical study revealed the induction of RIP3 and cIAP1/2 in the same motor neurons. These data suggest that transient normothermic ischaemia induces necroptosis, a potential factor in delayed motor neuron death, and that hypothermia may inhibit necroptosis.

Hypothermic Perfusion Hepatectomy for Unresectable Liver Cancer: A Single-Center Experience

Introduction: Despite technical advancements in liver surgery, resection of liver tumors that involve the hepatic veins adjacent to the vena cava or hepatic hilum remains technically challenging. We present our surgical techniques and the long-term outcome of 5 patients with conventionally unresectable tumors. Method: Between September 1999 and March 2016, we encountered 5 patients with conventionally unresectable tumors that were successfully treated by “ex-situ liver resection” and “in-situ & ante -situm hypothermic liver perfusion” under total vascular exclusion and venovenous bypass. Result: These approaches allowed complete tumor removal with vascular reconstruction under a bloodless operation field, while minimizing hepatic ischemic injury and preserving liver function. No perioperative mortalities occurred and postoperative complications were minimal. The postoperative survival periods were limited due to the advanced malignancies in our patients, but the survival benefit was encouraging. The median postoperative survival time was 29.1 months, with the longest survival period being nearly 10 years. Furthermore, these approaches improved the quality of life and provided our patients with an opportunity for additional treatment. Conclusion: Hypothermic perfusion hepatectomy is a realistic option for achieving surgical cure or significantly improved survival and quality of life in patients with tumors deemed unresectable using conventional normothermic hepatectomy. These approaches can overcome the limitations of the liver’s restricted normothermic ischemia tolerance or inaccessible tumor locations.

Topical hepatic hypothermia associated with ischemic preconditioning. Histopathological and biochemical analysis of ischemia reperfusion damage in a 24 hour model 1.

To develop a new 24 hour extended liver ischemia and reperfusion (LIR) model analyzing the late biochemical and histopathological results of the isolated and combined application of recognized hepatoprotective mechanisms. In addition, we used a new stratification with zoning to classify the histological lesion. A modified animal model of severe hepatic damage produced through 90 minutes of segmental ischemia (70% of the organ) and posterior observation for 24 hours of reperfusion, submitted to ischemic preconditioning (IPC) and topical hypothermia (TH) at 26ºC, in isolation or in combination, during the procedure. Data from intraoperative biometric parameters, besides of late biochemical markers and histopathological findings, both at 24 hours evolution time, were compared with control (C) and normothermic ischemia (NI) groups. All groups were homogeneous with respect to intraoperative physiological parameters. There were no losses once the model was stablished. Animals subjected to NI and IPC had worse biochemical (gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, direct bilirubin, and total bilirubin) and histopathological scores (modified Suzuki score) compared to those of control groups and groups with isolated or associated TH (p < 0.05). The new extended model demonstrates liver ischemia and reperfusion at 24 hour of evolution and, in this extreme scenario, only the groups subjected to topical hypothermia, combined with ischemic preconditioning or alone, had better outcomes than those subjected to only ischemic preconditioning and normothermic ischemia, reaching similar biochemical and histopathological scores to those of the control group.

Complex liver resection with in situ hypothermic portal perfusion

Introduction: Total vascular exclusion (TVE) of the liver might be required for complex liver resection. Standard TVE is contraindicated in some situations harboring a high risk of prolonged normothermic ischemia related morbidity. In situ hypothermic portal perfusion (HPP) can be used to overcome prolonged TVE consequences. Methods: This study is a prospective observational case series from January 2012 to December 2016, including patients with malignant tumors involving the vena cava and/or the hepatocaval confluence. Two surgical techniques were used: standard TVE with HPP and venovenous bypass and HPP with preservation of the caval flow using a temporary porto-caval shunt. Results: During the study period, 443 liver resections were performed in our department. Among them, fourteen (3.1%) were performed using HPP. Mean age was 55 years. Twelve (86%) patients had underlying liver disease. Mean tumor size was 87 mm (40-170mm). Mean operative time was 470 mn (360-580mn). Vascular reconstruction was performed in 4 (28.6%) patients. The 90-day mortality rate was 7.1% (1 patient). Four (28.6%) patients experienced grade III of Clavien-Dindo classification postoperative morbidity. No cases of postoperative liver failure or renal insufficiency were observed. Conclusion: HPP should be considered in patients requiring complex liver resections for centrally located tumors involving the hepatocaval confluence as it allows preservation of the future liver remnant function. Meanwhile, such resections should be indicated in selected patients with low comorbidity and good performance status because of a high morbidity rate.

Notch1 Mediates Preconditioning Protection Induced by GPER in Normotensive and Hypertensive Female Rat Hearts.

G protein-coupled estrogen receptor (GPER) is an estrogen receptor expressed in the cardiovascular system. G1, a selective GPER ligand, exerts cardiovascular effects through activation of the PI3K-Akt pathway and Notch signaling in normotensive animals. Here, we investigated whether the G1/GPER interaction is involved in the limitation of infarct size, and improvement of post-ischemic contractile function in female spontaneous hypertensive rat (SHR) hearts. In this model, we also studied Notch signaling and key components of survival pathway, namely PI3K-Akt, nitric oxide synthase (NOS) and mitochondrial K+-ATP (MitoKATP) channels. Rat hearts isolated from female SHR underwent 30 min of global, normothermic ischemia and 120 min of reperfusion. G1 (10 nM) alone or specific inhibitors of GPER, PI3K/NOS and MitoKATP channels co-infused with G1, just before I/R, were studied. The involvement of Notch1 was studied by Western blotting. Infarct size and left ventricular pressure were measured. To confirm endothelial-independent G1-induced protection by Notch signaling, H9c2 cells were studied with specific inhibitor, N-[N-(3,5 difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT, 5 μM), of this signaling. Using DAPT, we confirmed the involvement of G1/Notch signaling in limiting infarct size in heart of normotensive animals. In the hypertensive model, G1-induced reduction in infarct size and improvement of cardiac function were prevented by the inhibition of GPER, PI3K/NOS, and MitoKATP channels. The involvement of Notch was confirmed by western blot in the hypertensive model and by the specific inhibitor in the normotensive model and cardiac cell line. Our results suggest that GPERs play a pivotal role in mediating preconditioning cardioprotection in normotensive and hypertensive conditions. The G1-induced protection involves Notch1 and is able to activate the survival pathway in the presence of comorbidity. Several pathological conditions, including hypertension, reduce the efficacy of ischemic conditioning strategies. However, G1-induced protection can result in significant reduction of I/R injury also female in hypertensive animals. Further studies may ascertain the clinical translation of the present results.

A dose-response study of glutamate supplementation in isolated, perfused rat hearts undergoing ischaemia and cold cardioplegia.

Several studies have reported superior post-cardioplegic recovery after glutamate supplementation. The optimum dose of glutamate supplementation is unknown. The purpose of this study was to find the optimal protective concentration of glutamate supplementation in a model of ischaemia/cardioplegia and reperfusion. Isolated rat hearts (n = 77) were perfused with the Krebs-Henseleit buffer. After stabilization, the hearts were subjected to 25 min of normothermic ischaemia followed by a single 3-min infusion of cold (4-6 °C) St. Thomas' Hospital II cardioplegia and 87 min of cardioplegic ischaemic arrest and 60 min of reperfusion. Sodium-l-glutamate was added to the perfusate (control group had zero glutamate) in increasing concentrations (0.01, 0.1, 1, 10, 20, 30 and 100 mM) and given throughout perfusion. Corresponding concentrations were added to the cardioplegic solution. A balloon in the left ventricle inserted via the left atrium measured left ventricular pressures isometrically. Left ventricular developed pressure was calculated. Myocardial exchange of glucose and lactate was measured prior to ischaemia and during reperfusion. Myocardial content of glycogen and glutamate was measured at the end of reperfusion. During reperfusion left ventricular developed pressure increased (P < 0.0001) in groups supplemented with 0.1, 1.0, 10, 20 and 30 mM glutamate, whereas left ventricular end-diastolic pressure was attenuated (P = 0.008) when compared with the controls. No additional benefit on the continuous data left ventricular developed pressure and left ventricular end-diastolic pressure was observed with glutamate concentrations above 1 mM. Onset of LV pressure rise during the period of ischaemia was delayed by 100 mM of glutamate (P = 0.02). Myocardial content of glutamate was increased in a dose-related manner in Groups 10, 20, 30 and 100 compared with the control hearts (P < 0.0001). Glycogen was increased in the hearts supplemented with 100 mM of glutamate (P = 0.02). Even low concentrations of l-glutamate improved postischaemic and post-cardioplegic heart function and 1 mM seems to be optimal.

Efficacy of esmolol cardioplegia during hypothermic ischaemia

Cardioplegic arrest using a polarizing solution has been shown to have beneficial advantages for cardioprotection compared with depolarizing (potassium-based) arrest; most studies, however, have looked at normothermic ischaemia with short infusion intervals (every 10-15 min). This study examines the protective efficacy of an esmolol-based cardioplegia during hypothermic arrest, together with a prolonged infusion interval (30 min) for increased clinical feasibility. Isolated Langendorff-perfused hearts were subjected to arrest with St Thomas' Hospital cardioplegia (STH2), or esmolol cardioplegia (single- or multidose infusion at 32°C) for 60-min, 90-min or 120-min global ischaemia at 32°C, and recovery of function (left ventricular developed pressure) measured. A further study examined the protective efficacy of multidose esmolol cardioplegia compared with hypothermia alone at temperatures of 20°C, 28°C and 32°C compared with 37°C for 120 min of ischaemia. Esmolol cardioplegic arrest with multidose infusion at 32°C significantly improved recovery of function (left ventricular developed pressure) compared with 32°C STH2, at each ischaemic duration (88 ± 3 vs 66 ± 3% at 60 min, 82 ± 3 vs 51 ± 3% at 90 min and 73 ± 6 vs 49 ± 4% at 120 min; P < 0.05). At various hypothermic temperatures, esmolol cardioplegia significantly improved protection compared with hypothermia alone (88 ± 4%, 88 ± 3% and 72 ± 3% vs 60 ± 3%, 30 ± 2% and 15 ± 1% at 20°C, 28°C and 32°C, respectively; P < 0.05); however, at 37°C, there was no difference in protection. Contracture during ischaemia mirrored the effects of left ventricular developed pressure recovery. Esmolol cardioplegia (a polarizing solution), used as a multidose infusion during hypothermia, significantly improved cardioprotection compared with the depolarizing STH2. An increased infusion interval of 30 min indicates improved clinical feasibility.

PRELIMINARY RESULTS OF TOPICAL HEPATIC HYPOTHERMIA IN A MODEL OF LIVER ISCHEMIA/REPERFUSION INJURY IN RATS.

Ischemia/reperfusion causes organ damage but it is mandatory in hepatic transplantation, trauma and other complex liver surgeries, when Pringle maneuver is applied to minimize bleeding during these procedures. It is well known that liver ischemia/reperfusion leads to microcirculatory disturbance and cellular injury. In this setting hypothermia is known to reduce oxygen demand, lowering intracellular metabolism. To evaluate the effects of hypothermia in liver ischemia/reperfusion injury, using a new model of topic isolated liver hypothermia. We used male Wistar rats weighting about 250 grams, kept in ad libitum feeding regime and randomly divided into two groups of nine animals: 1) Normothermic group, rats were submitted to normothermic ischemia of the median and left hepatic lobes, with subsequent resection of right and caudate lobes during liver reperfusion; and 2) Hypothermic group, rats were submitted to liver ischemia under hypothermia at 10°C. Liver ischemia was performed for 45 minutes. The animals were euthanized 48 hours after liver reperfusion for blood and liver tissue sampling. The transaminases analyses showed a significant decrease of AST and ALT in Hypothermic group (P<0.01) compared to Normothermic group (1403±1234 x 454±213 and 730±680 x 271±211 U/L, respectively). Histology showed severe necrosis in 50% and mild necrosis in 50% of cases in Normothermic group, but severe necrosis in 10% and mild or absent necrosis 90% of the cases in hypothermic group. A simplified model of liver ischemia/reperfusion that simulates orthotopic liver autotransplantion was demonstrated. Topical hypothermia of isolated hepatic lobules showed liver protection, being a viable and practical method for any kind of in vivo liver preservation study.

Early experience with hypothermic machine perfusion of living donor kidneys - a retrospective study.

Although hypothermic machine perfusion (HMP) has been shown to be beneficial to deceased donor kidneys, the effect of HMP on living donor kidneys (LDK) is unknown. LDK are subjected to minutes of normothermic ischemia at the time of recovery. Comparison of 16 LDK preserved by HMP with 16 LDK preserved by static cold storage (SCS). Outcomes of interest are resistive indices (RI), both while on HMP and postoperatively, and creatinine clearance (CrCl). Injury markers NGAL and LDH were seen in the perfusate of LDK in amounts similar to what is found for donation after neurological determination of death kidneys. Compared to SCS kidneys, CrCl was significantly higher in the HMP group from days 2 through 7 post-transplant [ie: day 7 (78.8 ± 5.4 vs. 54.0 ± 4.6 ml/min, P = 0.005)]. CrCl at 1 year was higher in the HMP group (81.2 ± 5.8 vs. 70.0 ± 5.3 ml/min, P = 0.03). Early post-transplant RI was significantly lower in the HMP group (0.61 ± 0.02 vs. 0.71 ± 0.02, P < 0.0001). Our data support the assertion that injury does occur during LDK procurement and suggest that some of this injury may be reversed with HMP, resulting in more favorable early RI and graft function compared to SCS kidneys.

Age-related differences of intraischemic gap junction uncoupling in hearts during ischemia

ObjectiveMyocardial ischemia leads to energetic, morphologic, metabolic, and functional alterations. To evaluate differences in ischemia tolerance between neonatal and adult hearts, we investigated gap junction uncoupling (GJU) and its correlation to myocardial intracellular edema formation during normothermic ischemia. MethodsHearts of landrace piglets (neonates, 7.4 ± 1.9 days of age, body weight 2.9 ± 0.5 kg, n = 5 and adults, 84 ± 9 days of age, body weight 30.5 ± 3.9 kg, n = 5) were investigated. After we harvested the hearts, the bioelectrical impedance spectra were measured continuously during normothermic global ischemia (35°C). Spectra of the dielectric permittivity, ε′(frequency), and conductivity, σ(frequency), were calculated from the impedance measurements, and GJU was identified in the sigmoidal time course of ε′ (13 kHz). The extracellular volume was estimated by the ratio σ (100Hz)/σ (1MHz). Dielectric data were correlated with electron-microscopical images. ResultsIntraischemic GJU was observed in neonates after 54 ± 9 minutes of ischemia and thus significantly earlier than in adults (90 ± 7 minutes, P < .05). A more than 20% increase of intercalated water was found in tissue samples of neonates after 20 ± 2 minutes, in contrast to adults after 137 ± 8 minutes (P < .05). ConclusionsIntraischemic formation of edema and earlier GJU indicate faster intraischemic changes in neonates compared with adults. Intraischemic GJU and determination of intracellular water shifts are an experimental approach to establish the period of life-threatening damage. Because both parameters are linked and occur significantly earlier in neonates, they distinctly demonstrate the lower ischemia tolerance of neonatal hearts as both events interact.

Remote ischemic perconditioning attenuates acute inflammation of experimental musculocutaneous flaps following ischemia-reperfusion injury.

In free flap reconstruction and replantation surgery, prolonged ischemia time may lead to flap or replantation failure. The aim of the study was to investigate the effects of hypothermic flap ischemia or remote ischemic perconditioning (RIPER) during normothermic ischemia on acute inflammation of musculocutaneous flaps subjected to ischemia-reperfusion injury. In 24 pigs, a musculocutaneous latissimus dorsi flap was dissected and subjected to 4 hours of arterial ischemia and 7 hours of reperfusion. The animals were allocated into two experimental groups: hypothermic flap ischemia at 4°C (n=8) or normothermic flap ischemia with RIPER (n=8), and one control group with normothermic flap ischemia (n=8). The hypothermic ischemic flaps were cooled in a basin with fresh water and ice. RIPER was initiated 1hour before reperfusion, by inducing three 10min cycles of hind limb ischemia with a tourniquet, each separated by 10min of reperfusion. Acute inflammation was described by inflammatory cytokine secretion (IL-1β, IL-6, IL-10, IL-12p40, and TNF-α) from the flap during reperfusion, and by quantitative determination of macrophages in flap biopsies of dermis, subcutaneous tissue, and skeletal muscle following reperfusion. No significant differences were found between normothermic and hypothermic flap ischemia in inflammatory cytokine secretion. However, the IL-6 secretion was significantly reduced in the RIPER group compared with the control group at 5hours of reperfusion (P=0.036), and in the RIPER group compared with the hypothermic ischemia group at 3 (P=0 0.0063), 5 (P=0.0026), and 7hours of reperfusion (P=0.028). The IL-12p40 secretion was significantly reduced in the RIPER group compared with the control group (P=0.0054) as well as the hypothermic ischemia group (P=0.028) at 5hours of reperfusion. No significant difference was found among groups in macrophage infiltration. RIPER reduced IL-6 and IL-12p40 secretion during reperfusion of porcine musculocutaneous flaps, when compared with hypothermic ischemic flaps and normothermic ischemic flaps without RIPER. © 2016 Wiley Periodicals, Inc. Microsurgery 37:148-155, 2017.

0110 : Experimental cerebral ischemia in rats increases myocardial vulnerability to ischemia-reperfusion injury ex vivo

For years, the relationship between cardiac and neurological ischemic events has been mainly attributed to overlapping pathophysiological mechanisms and common risk factors. However, acute stroke may induce dramatic alterations of cardiovascular function. The aim of this work was to evaluate how prior cerebrovascular lesions affect myocardial function in vivo and ex vivo , as well as myocardial vulnerability to ischemic injury. Cerebral embolization was performed in adult Wistar male rats by the injection of microspheres into the left internal carotid artery. Left ventricular function, investigated in vivo using echocardiography (1 hour, 24 hours and 7 days after the embolization), was not significantly impaired; however, the heart rate was significantly increased in the stroke group (+7.2%). Epinephrine (E) and norepinephrine (NE) plasma levels increased in rats from the stroke group (E: 47.3±2.1 vs. 24.3±8.7 and NE: 22.7±4.2 vs. 10.9±3.7). One hour after stroke or sham embolization, hearts were isolated and perfused ex vivo in the Langendorff mode. In hearts from the stroke group, the baseline left ventricular developed pressure was diminished (-11%); moreover, a greater myocardial vulnerability to ischemic injury was observed, with impaired coronary flow recovery after 40 minutes of total global normothermic ischemia. Our study provides original exciting data indicating that myocardial vulnerability to ischemia can be worsened by prior ischemic stroke, a situation that does not agree with the concept of remote preconditioning. The underlying molecular mechanisms of the stroke-induced myocardial alterations after cerebral embolization remain to be established, insofar as they may involve the sympathetic nervous system. The author hereby declares no conflict of interest

Gap Junction Regulation and Edema Formation in Neonatal and Adult Hearts

Objectives: In congenital heart surgery, ischemia-induced myocardial edema formation often influences postoperative care, especially after long lasting ischemic periods, since delayed sternal closure is necessary. To evaluate differences in ischemic processes between neonatal and adult hearts we investigated gap junction closure (GJC) and edema formation during normothermic ischemia (35°C) using bioelectrical impedance spectroscopy.

Hypothermia is better than ischemic preconditioning for preventing early hepatic ischemia/reperfusion in rats

Background. Topical hypothermia (TH) and ischemic preconditioning (IPC) are used to decrease I/R injury. The efficacy of isolated or combined use of TH and IPC in the liver regarding inflammation and cytoprotection in early ischemia/reperfusion (I/R) injury needs to be evaluated.Material and methods. Wistar rats underwent 70% liver ischemia for 90 min followed by 120 min of reperfusion. Livers of animals allocated in the sham, normothermic ischemia (NI), IPC, TH, and TH+IPC groups were collected for molecular analyses by ELISA and Western blot, aiming to compare proinflammatory, anti-inflammatory, and antioxidant profiles.Results. Compared with NI, TH presented decreased tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 and IL-12 concentrations and increased IL-10 levels. TH animals displayed lower inducible nitric oxide synthase (iNOS) and higher endothelial nitric oxide synthase (eNOS) expressions. NAD(P)H-quinone oxidoreductase-1(NQO1) expression was also lower with TH. Isolated IPC and NI were similar regarding all these markers. TH+IPC was associated with decreased IL-12 concentration and reduced iNOS and NQO1 expressions, similarly to isolated TH. Expression of Kelch-like ECH-associated protein (Keap)-1 was increased and expression of nuclear and cytosolic nuclear erythroid 2-related factor 2 (Nrf2) was decreased with TH+IPC vs. NI.Conclusion. TH was the most effective method of protection against early I/R injury. Isolated IPC entailed triggering of second-line antioxidant defense enzymes. Combined TH+IPC seemed to confer no additional advantage over isolated TH in relation to the inflammatory process, but had the advantage of completely avoid second-line antioxidant defense enzymes.