Period Of Ischemia Research Articles

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

Remote ischemic preconditioning promotes cardioprotection through a central nervous system dependent mechanism

Remote ischemic preconditioning (rIPC) is the phenomenon of brief periods of ischemia and reperfusion applied to a distant organ promote cardioprotection against a subsequent ischemia/reperfusion injury (I/R). Although a neurohumoral hypothesis has been proposed, the cardioprotective mechanisms of rIPC have not been fully elucidated. We investigated whether rIPC could induce cardioprotection in a pithed Wistar rat model hypothesizing that the absence of central nervous system (CNS) structures would prevent its cardioprotective effect. rIPC protocol consisting of 3 cycles of 4 minutes of ischemia and 4 minutes of reperfusion was applied to the left posterior extremity of male Wistar rats. I/R was induced surgically by left coronary descending artery ligation for 30 minutes followed by 2h of reperfusion. Isolated rat hearts were perfused in a Langendorff system to measure coronary vascular resistance (CVR). Finally, the infarct size was evaluated by Evans blue and triphenyltetrazolium staining. rIPC reduced the infarct size of the ischemic hearts; however, this effect was abolished in pithed rats. rIPC also prevented the rise of CVR of ischemic hearts while there was no difference compared to pithed rats. We concluded that rIPC reduces the infarct size of hearts during I/R preserving the coronary artery tone through a CNS‐dependent mechanism.

Cardioprotective effects of redox regulator MnTnBuOE‐2‐PyP 5+ in myocardial ischemia/reperfusion injury

BackgroundMyocardial infarction is a leading cause of death worldwide and occurs due to decrease or complete blockage of coronary blood flow. Re‐establishment of blood flow after a brief period of ischemia is accompanied by exacerbation of the cardiomyocytes and its death, a phenomenon known as myocardial ischemia‐reperfusion (I/R) injury. Much evidence suggests that oxidative stress is involved in the pathogenesis of myocardial ischemia‐reperfusion injury and is potential target for therapeutic interventions. The aim of this study is to evaluate cardioprotective role of redox active Mn(III) N‐n‐butoxyethylpyridinium‐2‐yl)porphyrin, MnTnBuOE‐2‐PyP5+ (BMX‐001) in myocardial I/R injury. This drug is now in 4 Phase II clinical trials where the radioprotection of normal issue and anticancer efficacy have been evaluated.MethodsMyocardial I/R injury in mice C57BL/6 was induced by ligating left anterior descending artery for 1 hour followed by 24 hours of reperfusion. At the end of reperfusion, hearts were sectioned (1mm) and stained with 1% 2, 3, 5‐Triphenyltetrazolium chloride (TTC) to determine infarct size. Cardiac function was evaluated by M‐mode echocardiography at the end of the reperfusion. For in vitro model, H9c2 cells were subjected to hypoxia for 3 hours followed by 24 hours of reperfusion. Cell apoptosis was measured by TUNEL assay. Mitochondrial superoxide and Cardiolipin peroxidation were measured by using MitoSOX and 10‐N‐nonyl‐Acridin Orange, respectively.ResultsIn the current study, we demonstrated that treatment with MnTnBuOE‐2‐PyP5+ (2mg/kg) 4 hours before I/R injury in mice ameliorate cardiac injury as evident by limited infarct size and improved cardiac function (P<0.05). Consistently, MnTnBuOE‐2‐PyP5+ improved mitochondrial SOD2 expression (P<0.05) and reduced cardiomyocytes apoptosis in mice heart (P<0.05). In our in vitro model of hypoxia/reoxygenation injury in H9c2 cells, treatment with MnTnBuOE‐2‐PyP5+ (10µM) 24 hours before hypoxia/ reoxygenation injury decreased cardiolipin peroxidation, mitochondrial superoxide level and 4‐HNE adducted proteins (P<0.01). Additionally we explored role of MnTnBuOE‐2‐PyP5+ on cell apoptosis following ischemia/reperfusion injury in H9c2 cells. Redox regulator significantly reduced TUNEL positive cells following I/R injury indicating reduced cardiomyocytes apoptosis (p<0.05). In‐vitro, redox‐active Mn porphyrin improved cell viability and lactate dehydrogenase release following hypoxia/reoxygenation in H9c2 cells (p<0.05).ConclusionThis result provided evidence that MnTnBuOE‐2‐PyP5+ elicited cytoprotective effects through reduction of mitochondrial oxidative stress and cardiomyocyte apoptosis suggesting prospective therapeutics in myocardial I/R Injury.

Critical warm ischemia time point for cardiac donation after circulatory death

Donation after circulatory death (DCD) represents a promising opportunity to overcome the relative shortage of donors for heart transplantation. However, the necessary period of warm ischemia is a concern. This study aims to determine the critical warm ischemia time based on in vivo biochemical changes. Sixteen DCD non‐cardiac donors, without cardiovascular disease, underwent serial endomyocardial biopsies immediately before withdrawal of life‐sustaining therapy (WLST), at circulatory arrest (CA) and every 2 min thereafter. Samples were processed into representative pools to assess calcium homeostasis, mitochondrial function and cellular viability. Compared to baseline, no significant deterioration was observed in any studied parameter at the time of CA (median: 9 min; IQR: 7–13 min; range: 4–19 min). Ten min after CA, phosphorylation of cAMP‐dependent protein kinase‐A on Thr197 and SERCA2 decreased markedly; and parallelly, mitochondrial complex II and IV activities decreased, and caspase 3/7 activity raised significantly. These results did not differ when donors with higher WLST to CA times (≥9 min) were analyzed separately. In human cardiomyocytes, the period from WLST to CA and the first 10 min after CA were not associated with a significant compromise in cellular function or viability. These findings may help to incorporate DCD into heart transplant programs.

Weddell seals (Leptonychotes weddellii) respond less to inflammatory stimuli to evade ischemic‐reperfusion injury

During diving, Weddell seals (Leptonychotes weddellii) experience repeated hypoxemia. To mediate hypoxic tissue damage, Weddell seals employ vasoconstriction favoring vital organs (e.g. brain, lung, adrenals) resulting in reduced oxygen delivery to visceral and peripheral tissues. Weddell seals therefore experience periods of local ischemia associated with vasoconstriction followed by reoxygenation and return of perfusion at the surface. Ischemia‐reperfusion injury (IRI) is the expected result from reduced blood flow and localized hypoxia, followed by reoxygenation and reperfusion. A typical human response to IRI induces inflammation. We studied the inflammatory response of Weddell seals to determine whether its downregulation could be a mechanism by which seals avoid hypoxic and IRI injury. Weddell seal white blood cells (n=12) demonstrate a reduced immune reaction (mean ± stdev 247± 200 fold induction of IL‐6 from baseline) compared to human monocytes (n=6 replicates, THP‐1 cells 3091 ± 1126 fold IL‐6 induction, t‐test p<0.0001) when exposed to an inflammatory stimulus in vitro (10 ng/mL lipopolysaccharide). To evaluate species differences in gene pathways associated with the immune response, we conducted RNA‐seq and identified distinct transcriptomic profiles between species. We next evaluated the inflammatory response of Weddell seal primary cells (monocytes and pulmonary endothelial cells) resulting from hypoxia and reoxygenation. By simulating this natural stressor, we aim to discover whether the response of immune cells is an important component of molecular protection against IRI inflammatory sequelae in diving seals.

Normothermic ex situ pancreas perfusion for the preservation of porcine pancreas grafts

Pancreas transplantation improves and extends the life of patients with insulin-dependent diabetes. Pancreata from extended criteria donors have been increasingly used due to the scarcity of available grafts. Normothermic ex situ pancreas perfusion (NESPP) can keep grafts metabolically active, potentially allowing for assessment and organ repair, and could improve outcomes of marginal grafts. A novel NESPP technique was developed and tested. Porcine pancreata were removed after a short period of warm ischemia and subjected to 6 h of NESPP. Perfusion parameters, potential graft assessment markers and graft injury were measured. Next, pancreata subjected to 3 h of NESPP were transplanted and animals were followed for up to 3 days. Graft function and injury post-transplantation were evaluated. Using this novel system of perfusion, pancreata were perfused for an extended period of time with minimal edema. Histology at the end of perfusion showed intact islet cells with only mild signs of tissue injury. NESPP transplanted grafts showed immediate function after transplantation, with glucose levels in normal range. NESPP maintains a physiologic environment and excellent graft function without causing significant graft injury. Porcine pancreas transplantation is feasible and allows for in vivo graft assessment of pancreas function and injury after NESPP.

Hypoxic Preconditioning Attenuates Ischemia‐Reperfusion Injury in Older Adults

Sudden restoration of blood flow to an ischemic vessel paradoxically damages endothelial cells. In young healthy adults, ischemic preconditioning, caused by repeated periods of brief ischemia induced by local cuff inflation prior to reperfusion, attenuates endothelial dysfunction following an ischemia‐reperfusion injury. However, ischemic preconditioning does not consistently protect against ischemia‐reperfusion injury in older adults. Intermittent systemic hypoxemia, induced via brief bouts of breathing low levels of oxygen, attenuates endothelial dysfunction following an ischemia‐reperfusion injury in young healthy adults. Therefore, the aim of the present study was to determine whether intermittent hypoxia protects against ischemia‐reperfusion injury in older adults. Eleven older adults (4 women, age: 57±9 years, height: 173±8 cm, body weight: 76±13 kg) visited the laboratory on two separate occasions. Endothelium‐dependent vasodilation was assessed by brachial artery flow‐mediated dilation using a semiautomated diagnostic ultrasound system before and after 20 minutes of blood flow occlusion to induce an ischemia‐reperfusion injury. Blood flow occlusion was preceded by either intermittent hypoxia (IH), consisting of three 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with 4‐min room air cycles, or intermittent normoxia (IN), consisting of three 4‐min normoxic cycles separated by 4‐min room air cycles. Intermittent hypoxia resulted in an arterial oxygen saturation of 80±2%, which corresponded to oxygen levels of 11.6±1.0%. When preceded by intermittent normoxia, blood flow occlusion reduced flow‐mediated dilation by 4.0±2.6% (6.4±1.7 to 2.4±1.8%). In contrast, flow‐mediated dilation was reduced by 1.8±1.5% when blood flow occlusion was preceded by intermittent hypoxia (5.6±1.8 to 3.7±2.4%: p=0.05). Intermittent hypoxia increased cardiac output (5.2±1.5 to 5.7±1.8 L·min‐1, p=0.03) mainly due to an increase in heart rate (61±12 to 69±10 bpm, p<0.01). In conclusion, hypoxic preconditioning attenuated the reduction in flow‐mediated dilation induced by blood flow occlusion in older adults. Thus, exposure to intermittent hypoxia represents a potential strategy to protect against ischemia‐reperfusion injury in populations at risk for ischemic events.

Effect of cardioplegic arrest and reperfusion on left andright ventricular proteome/phosphoproteome in patients undergoing surgery for coronary or aortic valve disease.

Our earlier work has shown inter-disease and intra-disease differences in the cardiac proteome between right (RV) and left (LV) ventricles of patients with aortic valve stenosis (AVS) or coronary artery disease (CAD). Whether disease remodeling also affects acute changes occuring in the proteome during surgical intervention is unknown. This study investigated the effects of cardioplegic arrest on cardiac proteins/phosphoproteins in LV and RV of CAD (n=6) and AVS (n=6) patients undergoing cardiac surgery. LV and RV biopsies were collected during surgery before ischemic cold blood cardioplegic arrest (pre) and 20 min after reperfusion (post). Tissues were snap frozen, proteins extracted, and the extracts were used for proteomic and phosphoproteomic analysis using Tandem Mass Tag (TMT) analysis. The results were analysed using QuickGO and Ingenuity Pathway Analysis softwares. For each comparision, our proteomic analysis identified more than 3,000 proteins which could be detected in both the pre and Post samples. Cardioplegic arrest and reperfusion were associated with significant differential expression of 24 (LV) and 120 (RV) proteins in the CAD patients, which were linked to mitochondrial function, inflammation and cardiac contraction. By contrast, AVS patients showed differential expression of only 3 LV proteins and 2 RV proteins, despite a significantly longer duration of ischaemic cardioplegic arrest. The relative expression of 41 phosphoproteins was significantly altered in CAD patients, with 18 phosphoproteins showing altered expression in AVS patients. Inflammatory pathways were implicated in the changes in phosphoprotein expression in both groups. Inter-disease comparison for the same ventricular chamber at both timepoints revealed differences relating to inflammation and adrenergic and calcium signalling. In conclusion, the present study found that ischemic arrest and reperfusion trigger different changes in the proteomes and phosphoproteomes of LV and RV of CAD and AVS patients undergoing surgery, with markedly more changes in CAD patients despite a significantly shorter ischaemic period.

Mapping the Diversity of the Innate Immune System over Time After Left Lung Transplantation in Mice

Purpose Following lung transplantation, the surgical intervention with ischemic periods and the foreign allograft are the first to trigger a response from the innate immune system, which represents the primary nonspecific defense against “non-self”. In this study, we investigated the diversity of the emerging innate immune cells and the timing of their appearance in a murine orthotopic left lung transplant model. Methods Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6). All mice received daily immunosuppression of 10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone. Serial sacrifice was performed at day 1, 7 and 35 post-transplantation (n=6 at each time point for each group). Left transplanted lungs were harvested, single cell suspension was made and absolute numbers of innate immune cells were quantified by flow cytometry. Results Following transplantation, isografts and allografts showed an increase in neutrophil (p=0.017) and tissue resident NK cell (p=0.002) numbers at day 1, and in eosinophil (p=0.002) numbers at day 7 post-lung transplant. Granulocytes demonstrated similar patterns in isografts and allografts. Allografts demonstrated an increase in antigen presenting cells i.e. patrolling monocytes (Ly6C-) (p=0.002), interstitial macrophages (p<0.0001) and dendritic cells (p=0.005) at day 7 compared to isografts. Classical NK cells were increased in allografts at day 7 versus isografts (p=0.03). All cell numbers returned to baseline isograft values at day 35 post-transplant, except for dendritic cells (p=0.0045). Conclusion Diverse innate immune cell types show different patterns over time post-lung transplantation depending on transplant induced ischemia-reperfusion injury and/or alloimmune response against the foreign graft. These differences may be very important to dissect the various pathophysiological processes triggered by lung transplantation. Following lung transplantation, the surgical intervention with ischemic periods and the foreign allograft are the first to trigger a response from the innate immune system, which represents the primary nonspecific defense against “non-self”. In this study, we investigated the diversity of the emerging innate immune cells and the timing of their appearance in a murine orthotopic left lung transplant model. Mouse orthotopic left lung transplantation was performed in isografts (C57BL/6 to C57BL/6) and allografts (Balbc to C57BL/6). All mice received daily immunosuppression of 10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone. Serial sacrifice was performed at day 1, 7 and 35 post-transplantation (n=6 at each time point for each group). Left transplanted lungs were harvested, single cell suspension was made and absolute numbers of innate immune cells were quantified by flow cytometry. Following transplantation, isografts and allografts showed an increase in neutrophil (p=0.017) and tissue resident NK cell (p=0.002) numbers at day 1, and in eosinophil (p=0.002) numbers at day 7 post-lung transplant. Granulocytes demonstrated similar patterns in isografts and allografts. Allografts demonstrated an increase in antigen presenting cells i.e. patrolling monocytes (Ly6C-) (p=0.002), interstitial macrophages (p<0.0001) and dendritic cells (p=0.005) at day 7 compared to isografts. Classical NK cells were increased in allografts at day 7 versus isografts (p=0.03). All cell numbers returned to baseline isograft values at day 35 post-transplant, except for dendritic cells (p=0.0045). Diverse innate immune cell types show different patterns over time post-lung transplantation depending on transplant induced ischemia-reperfusion injury and/or alloimmune response against the foreign graft. These differences may be very important to dissect the various pathophysiological processes triggered by lung transplantation.

Lipopolysaccharide Pretreatment Prevents Medullary Vascular Congestion following Renal Ischemia by Limiting Early Reperfusion of the Medullary Circulation.

Vascular congestion of the renal medulla-trapped red blood cells in the medullary microvasculature-is a hallmark finding at autopsy in patients with ischemic acute tubular necrosis. Despite this, the pathogenesis of vascular congestion is not well defined. In this study, to investigate the pathogenesis of vascular congestion and its role in promoting renal injury, we assessed renal vascular congestion and tubular injury after ischemia reperfusion in rats pretreated with low-dose LPS or saline (control). We used laser Doppler flowmetry to determine whether pretreatment with low-dose LPS prevented vascular congestion by altering renal hemodynamics during reperfusion. We found that vascular congestion originated during the ischemic period in the renal venous circulation. In control animals, the return of blood flow was followed by the development of congestion in the capillary plexus of the outer medulla and severe tubular injury early in reperfusion. Laser Doppler flowmetry indicated that blood flow returned rapidly to the medulla, several minutes before recovery of full cortical perfusion. In contrast, LPS pretreatment prevented both the formation of medullary congestion and its associated tubular injury. Laser Doppler flowmetry in LPS-pretreated rats suggested that limiting early reperfusion of the medulla facilitated this protective effect, because it allowed cortical perfusion to recover and clear congestion from the large cortical veins, which also drain the medulla. Blockage of the renal venous vessels and a mismatch in the timing of cortical and medullary reperfusion results in congestion of the outer medulla's capillary plexus and promotes early tubular injury after renal ischemia. These findings indicate that hemodynamics during reperfusion contribute to the renal medulla's susceptibility to ischemic injury.

Cytosolic DNA Sensors as a New Pharmacological Target in EVLP

<h3>Purpose</h3> Innate immunity is a key component of the inflammatory responses ensuing ischemia/reperfusion (I/R) injury. Cell damage and death caused by I/R promotes the release of danger-associated molecular patterns (DAMPs) recognized by specific pattern recognition receptors (PRRs). DNA released from nucleus and mitochondria of injured cells is a known DAMP, which sustains inflammation and cell death when recognized by specific PRRs like cytosolic DNA (cDNA) sensors cGAS and AIM2. The cGAS/STING pathway induces the production of type I interferons (IFNs) and IFN stimulated genes (ISGs). The role of cDNA sensors in reperfused lung is currently not characterized. Thus, using the rat <i>ex vivo</i> lung perfusion (EVLP) and lung transplantation (LTx) models, we begin to investigate the role of cGAS/STING in the early inflammatory events of lung I/R. <h3>Methods</h3> Heart-Lung blocks from male Sprague-Dawley rats were cannulated in the pulmonary artery and left atrium and assigned to four groups (n=5/6 rats/group). Cold ischemia (CI): under mechanical ventilation, the lungs were flushed with 4°C Perfadex. The heart-lung block was stored inflated (FiO2 0.5) for 7h in 4°C Perfadex. Warm ischemia (WI): After cannulation, the lungs were deflated for 1h at room temperature, flushed and stored as above for 6h. CI+EVLP: lungs were harvested as above, stored at 4°C for 2h, followed by 3-hour EVLP, and stored again (2h) in 4°C Perfadex. WI+EVLP: after 1 hour WI, lungs were flushed and stored at 4°C for 1 hour, followed by 3h EVLP, and stored again (2h) in 4°C Perfadex. Right lungs were used as controls and left lungs were transplanted and sampled 2h after LTx. We analyzed by RT-PCR and western blot the expression of the cytosolic DNA sensors cGAS, STING and AIM2 and by RT-PCR the expression of the downstream gene <i>Ifnb</i>, and the ISGs <i>Ifit1</i> and <i>Isg15</i> in whole lung tissue<i>.</i> <h3>Results</h3> cGAS, STING and AIM2 proteins are expressed in lung after the ischemic period. Their mRNA expression is strongly induced by the reperfusion (EVLP, LTx or EVLP+LTx) regardless of the conditions of preservation (CI or WI). We observed a similar behavior for the mRNA expression of <i>Ifnb</i> and the ISGs <i>Ifit1</i> and <i>Isg15</i>. <h3>Conclusion</h3> These preliminary data provide evidence that cytosolic DNA sensors, especially the cGAS-STING axis, may play a prominent role in the early inflammation of the reperfused lung regardless of the preservation conditions.

Crystalloid Machine Perfusion with the Novel HTK-N Preservation Solution Reduces Microvascular Dysfunction in Circulatory Death Hearts

<h3>Purpose</h3> Microvascular dysfunction (MVD) in cardiac allografts is associated with allograft vasculopathy, graft failure, and death after heart transplantation. Prolonged ischemia and pronounced ischemia/reperfusion injury promote MVD. Circulatory death (CD) donor hearts are exposed to a warm ischemic period followed by machine (re)perfusion (MP) with warm blood. The novel histidine-tryptophane-ketoglutarate(HTK)-N solution has been developed to improve myocardial contractility and preserve the vascular viability of donor hearts. The impact of MP with hypothermic, oxygenated HTK-N solution on microvascular function has not been investigated yet. <h3>Methods</h3> In a pig model of CD, the microvascular function of hearts was either assessed for 60 min immediately after harvesting (CD-group) or hearts were maintained by MP with normothermic blood (CD-B group), hypothermic HTK (CD-HTK group), or HTK-N (CD-HTK-N group) solution for 4 h before microvascular evaluation (all N=5 per group). We measured microvascular blood flow (LDP) by a Laser-Doppler-Perfusion needle probe in the left ventricular (LV) wall in dependence of a balloon-induced LV-preload to evaluate the microvascular function. We analyzed the Immunoreactivity of intracellular (ICAM), vascular (VCAM), and platelet-endothelial (PECAM) cell adhesion molecules, as well as endothelial nitric oxide synthase (eNOS) of arterioles, and profiled the expression of 84 genes. We constructed gene expression networks using the Boruta machine learning algorithm for feature selection. <h3>Results</h3> Relative LDP (rLDP) was majorly improved by HTK-N perfusion (1.16±0.06) compared with the CD-group (0.96±0.07, p=0.142), CD-B- (0.86±0.07, p=0.016) and CD-HTK-group (0.92±0.04, p=0.065). After MP with HTK-N, the expression of endothelial viability marker eNOS majorly increased, and the expression of endothelial injury markers ICAM, VCAM, and PECAM majorly decreased. The groups CD and CD-B showed fragmented gene networks with heterogenous gene correlation. After HTK and HTK-N perfusion, groups showed centralized networks with homogenous correlation and majorly reconstituted cluster regions. <h3>Conclusion</h3> MP with HTK-N counteracts MVD, preserves the microvascular endothelium, and reduces the expression of cell adhesion molecules in the coronary microvasculature of CD-hearts.

Normothermic Regional Perfusion of Circulatory Dead Donor Hearts: Quick and Dirty or Slow and Steady?

<h3>Purpose</h3> We sought to compare two strategies for cardiac reanimation in normothermic regional perfusion (NRP) in a clinically relevant porcine model of donation after circulatory death (DCD). To this day, many different protocols for NRP have been described. <h3>Methods</h3> For NRP-strategy 1(30mProp), pigs (n=16) were anaesthetized with intravenous Propofol (3.5 mg/kg/h) and subjected to hypoxic circulatory arrest (CA) by halting mechanical ventilation. After an 8-minute warm ischemia period, donor hearts were reanimated by a 30-minute NRP-period with Propofol suspended. For NRP-strategy 2(60mSevo), pigs (n=10) were anaesthetized with inhaled Sevoflurane (3.5%) before institution of hypoxia induced CA. A 10-minute warm ischemia followed before 60 minutes NRP with Sevoflurane continued. Both groups were reperfused via central cannulation with the aortic arch vessels and infrarenal aorta occluded. NRP flow, oxygenation, and weaning were standardized between groups. We assessed hemodynamic function with invasive blood pressure and bi-ventricular pressure-volume recordings 30 minutes post-NRP. <h3>Results</h3> All hearts were successfully reanimated during NRP in both groups. During NRP, a significantly higher mean arterial blood pressure and a minor need for pressor support with norepinephrine were observed in the 60mSevo group compared to the 30mProp group. Cardiac output remained on baseline levels in both groups post-NRP. Arterial lactate was significantly lower in the 60mSevo group post-NRP compared to the 30-minProp group. Left ventricular end-systole elastance as a measure of contractility increased significantly from baseline to post-NRP by 0.5 mmHg/mL (95%CI 0.09 : 1.0) in the 60-minSevo group and increased non-significantly by 0.3 mmHg/mL (95%CI -0.04 : 0.7) in the 30minProp group, while right ventricular contractility increased non-significantly in both groups. <h3>Conclusion</h3> Hearts reperfused for 60 minutes combined with inhaled Sevoflurane showed superior contractile function compared to hearts subjected to 30-minute NRP and Propofol anaesthesia. The results indicate that longer NRP is beneficial in DCD heart donation.

Modification of Ischemia/Reperfusion-Induced Alterations in Subcellular Organelles by Ischemic Preconditioning.

It is now well established that ischemia/reperfusion (I/R) injury is associated with the compromised recovery of cardiac contractile function. Such an adverse effect of I/R injury in the heart is attributed to the development of oxidative stress and intracellular Ca2+-overload, which are known to induce remodeling of subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils. However, repeated episodes of brief periods of ischemia followed by reperfusion or ischemic preconditioning (IP) have been shown to improve cardiac function and exert cardioprotective actions against the adverse effects of prolonged I/R injury. This protective action of IP in attenuating myocardial damage and subcellular remodeling is likely to be due to marked reductions in the occurrence of oxidative stress and intracellular Ca2+-overload in cardiomyocytes. In addition, the beneficial actions of IP have been attributed to the depression of proteolytic activities and inflammatory levels of cytokines as well as the activation of the nuclear factor erythroid factor 2-mediated signal transduction pathway. Accordingly, this review is intended to describe some of the changes in subcellular organelles, which are induced in cardiomyocytes by I/R for the occurrence of oxidative stress and intracellular Ca2+-overload and highlight some of the mechanisms for explaining the cardioprotective effects of IP.

Structural and functional transformations of the brain in experimental mild traumatic brain injury

In mild traumatic brain injury, it is of interest to study neurode-generative conditions resulting from inflammatory changes in the nervous tissue. Purpose of the study: in the acute period in case of mild experimental traumatic brain injury, to reveal structural transformations of the nervous tissue of the brain. A modified model of a falling weight was used to reproduce of these trauma in adult rats. An immunohistochemical study of the brain with using rat-specific monoclonal antibodies to endothelin-1, glial fibrillar acidic protein, vimentin, and blood-brain barrier endothelial protein (SMI 71) was performed. It has been established that on the first day after injury in the cerebral cortex of animals, the spasm of blood vessels with capillary ischemia predominates. On day 8, there is an increase in the number of hyper- and hypochromic neurons, and after 14 days, restoration of the tone of the microcircu-latory bed is detected with signs of a violation of the permeability of the blood-brain barrier. A significant redistribution in the tissues of the cerebral hemispheres of glial elements containing acid glial protein and vimentin, as well as neurons producing endothelin-1, was noted. In the delayed post-traumatic period, compensatory reactions of the nervous tissue were revealed, which are characterized by the presence of morphological changes in neurons (an increase in the diameter and number of nucleoli in size) associated with intracellular regeneration, as well as the synthesis of various protein factors in them. Thus, the pathogenesis of mild experimental craniocerebral injury in the ischemic (1 day) and intermediate (8 days) periods is characterized by the presence of mild violations of the structural integrity of the nervous tissue of the brain. In the late post-traumatic period (14 days), neurons and astrocytes exhibit compensatory reactions.

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Prolonged Cold Ischemia Did Not Impair Mitochondrial Oxygen Consumption or Reactive Oxygen Species Production in Human Uterine Fundus and Horn Myometrium

Uterine transplantation may be a solution for infertility of uterine origin. Nevertheless, only three pregnancies with a live birth have so far been possible involving a uterine transplant from a brain-dead donor. Particularly, the impact of ischemia needs a better understanding. Analysis of mitochondrial respiration and production of reactive oxygen species (ROS) in muscle are of interest since they are pertinent markers of the harmful effects of ischemia. We therefore studied both uterine fundus and horn muscle mitochondrial use of oxygen and ROS production in eight women needing hysterectomy. High resolution respirometry and electron paramagnetic resonance allowed the determination of, respectively, myometrium oxidative capacity, hydrogen peroxide, mitochondrial free radical leak and superoxide anion production early (2 and 7 h) and late (24 h) following surgery. Mitochondrial oxygen consumption of the uterine fundus and horn tended to decrease with time but this was not statistically significant. Concerning ROS production, globally, we observed no significant change for H2O2, superoxide anion and free radical leak. In conclusion, a long period of cold ischemia did not impair myometrium mitochondrial respiration, only generating a transient H2O2 increase in uterine fundus. These data support that cold ischemia, even when prolonged, does not significantly alter uterine muscle oxidative capacity.

Temperature-Related Effects of Myocardial Protection Strategies in Swine Hearts after Prolonged Warm Ischemia.

Insufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after circulatory death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitably longer period of unprotected warm ischemia between circulatory arrest and graft procurement. In this scenario, we aim to improve heart preservation after a warm ischemic period of 20 min by testing different settings of myocardial protective strategies. Pig hearts were collected from a slaughterhouse and assigned to one of the five experimental groups: baseline (BL), cold cardioplegia (CC), cold cardioplegia + adenosine (CC-ADN), normothermic cardioplegia (NtC + CC) or normothermic cardioplegia + cold cardioplegia + adenosine (NtC-ADN + CC). After treatment, tissue biopsies were taken to assess mitochondrial morphology, antioxidant enzyme activity, lipid peroxidation and cytokine and chemokine expressions. NtC + CC treatment significantly prevented mitochondria swelling and mitochondrial cristae loss. Moreover, the antioxidant enzyme activity was lower in this group, as was lipid peroxidation, and the pro-inflammatory chemokine GM-CSF was diminished. Finally, we demonstrated that normothermic cardioplegia preserved mitochondria morphology, thus preventing oxidative stress and the subsequent inflammatory response. Therefore, normothermic cardioplegia is a better approach to preserve the heart after a warm ischemia period, with respect to cold cardioplegia, before transplantation.

Targeting Mitochondrial Large-Conductance Calcium-Activated Potassium Channel by Hydrogen Sulfide via Heme-Binding Site.

Reperfusion together with the preceding ischemic period results in serious damage to brain and heart tissues. Activation of potassium channels from the inner mitochondrial membrane leads to cytoprotection during such events. The mitochondrial large-conductance calcium-activated potassium channel (mitoBKCa) is one of these cytoprotective channels. It was previously shown that BKCa channels are blocked by hemin, which is present in excess during hemorrhage. In the experiments described in this work, we checked whether NaHS, known as a donor of gasotransmitter hydrogen sulfide (H2S), which can play an important role in cytoprotection, interacts with mitoBKCa channels. Indeed, using the biotin-switch method, it was found that mitoBKCa channels undergo S-sulfhydration in the presence of NaHS. Although patch-clamp experiments showed that NaHS has negligible effects on the activity of mitoBKCa channels, NaHS has been shown to almost fully activate hemin-inhibited mitoBKCa channels. The effects of NaHS were mimicked by imidazole, suggesting a common mechanism of activation of mitoBKCa channels inhibited by heme/hemin by molecules able to coordinate the iron ion of porphyrin. A set of absorption spectroscopy experiments with the 23 amino acid model peptides containing the heme-binding motif CXXCH suggested previously unrecognized roles of cysteines in heme binding. SIGNIFICANCE STATEMENT: The activity of mitochondrial channels including mitoBKCa seems to play a significant role in cytoprotection during ischemia/reperfusion. Hemin, which is present in excess during hemorrhage, can potentially bind to and inhibit mitoBKCa activity. We found that hydrogen sulfide does not affect mitoBKCa activity unless it is blocked by hemin. In this case, hydrogen sulfide activates hemin-inhibited mitoBKCa by binding to hemin iron. The hydrogen sulfide effect could be mimicked in patch-clamp experiments by imidazole probably acting by a similar mechanism.