Stage Of Reperfusion Research Articles

The Protective Effect of a Chimeric PSH Antioxidant Enzyme in Renal Ischemia–Reperfusion Injury

Abstract—A rapid increase in the concentration of free radicals and reactive oxygen species at the reperfusion stage is the most dangerous stage of ischemia–reperfusion injury. An avalanche-like increase in the level of reactive oxygen species and secondary products of free radical oxidation of biological macromolecules leads to the development of oxidative stress. The use of exogenous antioxidants can reduce the concentration of reactive oxygen species in the affected tissues and suppress or correct the course of oxidative stress; thus, it significantly reduces the severity of ischemia–reperfusion injury. Acute ischemic renal failure is one of the most important social problems in a comprehensive list of pathologies associated with ischemia–reperfusion. The nephroprotective effect of a chimeric PSH antioxidant enzyme that includes human peroxiredoxin 6 and Mn-containing superoxide dismutase of Escherichia coli has been shown on an animal model of bilateral ischemia–reperfusion renal injury. The recombinant chimeric PSH protein was able to neutralize a maximally possible wide range of reactive oxygen species due to the superoxide dismutase and peroxidase activities. It has been shown with histological, biochemical, and molecular biological methods that the preliminary administration of the chimeric PSH protein before ischemia–reperfusion significantly reduced the degree of renal tissue injury and led to a quick normalization of their structural and functional state. In addition, the administration of the PSH enzyme increased the survival of experimental animals by a factor of more than 1.5. The use of the recombinant chimeric PSH enzyme can be an effective approach in the prevention and treatment of renal ischemia–reperfusion injury, as well as for maintaining an isolated kidney during transplantation.

Abstract TP286: The Study of the Role and Mechanism of TRPM7 in Acute Cerebral Ischemia-Reperfusion Injury

Although the NMDA receptor was identified to mediate excitotoxicity for ischemia-reperfusion (IR) injury, its antagonists have not shown clinical efficacy. Unrelated to NMDA, the transient receptor potential cation channel, subfamily M, member 7 (TRPM7) has been reported to cause intracellular Ca 2+ overloading, oxidative stress and neuronal apoptosis. We postulated that TRPM7 could be a target for treatment of IR injury. To investigate the potential neuroprotective role of TRPM7 in IR injury, 1) qRT-PCR, Western blot and immuno-staining were used to identify the expression and localization of TRPM7 in normal rat forebrain cortex and cultured primary cortical neurons; 2) the oxygen glucose deprivation and reperfusion (OGD/R) model of primary cortical neurons was used to detect changes in TRPM7 expression and study the effect of two TRPM7 ion channel inhibitors, carvacrol and 2-Aminoethoxydiphenyl borate (2-APB), on neurons in OGD/R; 3) the rat middle cerebral artery (MCAO/R) model was used to study TRPM7 involvement and the effect of its inhibitor during the course of IR. Our results showed that: 1) TRPM7 was expressed in normal neurons and astrocytes mainly as a cell membrane protein in the rat cortex; 2) TRPM7 expression was enhanced in OGD/R treated neurons; treatment with carvacrol or 2-APB reversed the overexpression of TRPM7 in OGD/R neurons and reduced the level of Ca 2+ overload in neuronal IR injury and neuronal apoptosis; 3) TRPM7 expression was also augmented in rat cortical tissues during MCAO/R and peaked between 24-48 hours during the reperfusion stage; 4) treatment with TRPM7 inhibitors reversed TRPM7 overexpression, alleviated neuronal apoptosis, and improved neurobehavioral scores in MCAO/R rats. Our conclusions are: 1) TRPM7 is expressed in normal rat forebrain cortex and functions mainly as a cell membrane protein in neurons and astrocytes; 2) TRPM7 is overexpressed during cerebral ischemia-reperfusion in rats; 3) TRPM7 ion channel inhibitors carvacrol and 2-APB can alleviate OGD/R injury in neurons in vitro as well as cortical damage by MCAO/R in rat forebrain; 4) the mechanism of TRPM7 on neuronal injury in OGD/R and MCAO/R can contribute to its role in neuronal apoptosis and Ca 2+ influx toxicity.

Dynamic metabolomic analysis of intestinal ischemia-reperfusion injury in rats.

Intestinal ischemia-reperfusion injury (IIR) is a life-threatening abdominal emergency. Compared to traditional steady-state works, we profiled the blood of rats over 72 hr (15 time points) and examined dynamic changes in molecular pathways during IIR. Using a series of methods designed for dynamic datasets analysis (batch effects corrections, metabolomics data reduction, and different features selection), we identified 39 significant different metabolites and discovered the trends of these molecules. Four main patterns were uncovered by a longitudinal pattern recognition method. Furthermore, pathway networks were explored to uncover the possible mechanisms of IIR. We found that IIR is a complex physiological process involved in multiple pathways, such as biosynthesis of amino acids, 2-oxocarboxylic acid metabolism, arginine-related metabolism, and glutathione metabolism. Among which, metabolites related with phenylalanine tyrosine and tryptophan metabolism reached a peak during the early stage of reperfusion, while molecules in biosynthesis of unsaturated fatty acids metabolism declined. Our work provides a feasible scheme to understand dynamic molecule variation and will provide new explications about the effect of intestinal ischemia reperfusion from a dynamic perspective.

En-Bloc Simultaneous Heart-Liver Transplantation in Adult Patients.

Complexity of combined heart-liver transplantation has resulted in low adoption rates. We report a case series of adult patients receiving en-bloc heart-liver transplantation (HLTx), describe technical aspects, and discuss benefits of the technique. Retrospective review of patients receiving en-bloc HLTx over 18 months, with clinical follow up to 1 year. Primary outcomes included postoperative mortality and major complications. Secondary outcomes included 1-year survival, cardiac or hepatic allograft rejection, and infection. Five patients received en-bloc HLTx. Mean recipient age was 43 years (26-63), and 3 patients were male. Total operative time was 430 minutes (393-480), cold and warm ischemic times of 85 (32-136) and 37.5 (31-47) minutes. Hospital survival was 80%. One patient died on postoperative day 55 due to fungal sepsis. Major postoperative complications included prolonged mechanical ventilation in 2 of 5 patients (40%), and renal insufficiency requiring hemodialysis in 2 of 5 patients (40%). Among patients discharged from hospital 1-year survival was 100%, with no evidence of rejection or infectious complications. En-bloc HLTx technique is a safe and effective strategy, decreasing operative times, and allograft ischemic times, whereas offering protection of implanted allografts during early stages of reperfusion while patient is hemodynamically supported on cardiopulmonary bypass.

Intraoperative management and early post-operative outcomes of patients with coronary artery disease who underwent orthotopic liver transplantation

BackgroundCoronary artery disease (CAD) is frequently observed in aging end-stage liver disease (ESLD) patients who require orthotopic liver transplantation (OLT). This situation is challenging for both the patients and the medical staff. MethodsWe retrospectively studied the case records of 26 ESLD patients with CAD who underwent OLT with total clamping of the inferior vena cava between 2014 and 2018. We analyzed the details of the pre-operative evaluation, intraoperative anesthetic management and post-operative prognosis of these patients. ResultsAll patients tolerated the anhepatic stage well. Post-reperfusion syndrome (PRS) was observed in 13 patients (50%) and 2 of them were severe but corrected well. ST-segment depression was frequently observed during the anhepatic stage and reperfusion stage. No mortality due to cardiac-related events occurred among the patients during hospitalization. OLT with the modified piggyback technique could successfully be performed in ESLD patients with mild and moderate CAD. ConclusionsA thorough evaluation and optimization of recipients, strict monitoring and optimized management of circulation, knowledge of the complicated changes during OLT procedures, and strategies to ameliorate post-reperfusion syndrome favorite the outcomes.

Catestatin in defense of oxidative-stress-induced apoptosis: A novel mechanism by activating the beta2 adrenergic receptor and PKB/Akt pathway in ischemic-reperfused myocardium

Apoptosis induced by oxidative stress is one of the most important cardiomyocytes losses during ischemia-reperfusion (I/R). Catestatin (CST) has been demonstrated to have the anti-oxidative capacity in vitro. We hypothesized that CST intervention could reduce apoptosis of cardiomyocytes induced by oxidative stress in I/R. In Langendorff-perfused rat heart global I/R model, CST was introduced at the reperfusion stage. In comparison to the control group, CST led to preservation on activities of superoxide dismutase and glutathione peroxidase, improvement of hemodynamics, and reduced infarction area in reperfused myocardium. The protection of CST was also shown by less apoptotic cardiomyocytes in TUNEL staining, less caspase-3 activation, and increased phosphorylation of protein kinase B (PKB/Akt) in Western blot. To further demonstrate the benefits of CST and explore the possible underlying mechanism, H2O2-challenged primary-cultured neonatal rat cardiomyocytes were used to simulate the oxidative-stressed scenario. CST incubation with the H2O2-challenged cardiomyocytes led to reduction of apoptosis, which was demonstrated by less Hoechst 33342 positive staining of nuclei, less caspase-3 activation, and DNA fragmentation. The effect of CST was abrogated by pretreatment of the cardiomyocytes with the PI3K inhibitor LY294002. Furthermore, Akt activation and the anti-apoptosis effect of CST were abolished by pretreatment of the cardiomyocytes with β2 receptor inhibitor ICI118551. Thus, the salvage of oxidative-stress-induced apoptotic cardiomyocytes in I/R by CST might involve activation β2 receptor and regulation of PI3K/Akt signaling in reperfusion injury salvage kinase (RISK) pathway.

Characteristics of Changes in Inflammatory Cytokines as a Function of Hepatic Ischemia-Reperfusion Injury Stage in Mice.

Liver ischemia-reperfusion injury (IRI) can severely compromise the prognosis of patients receiving liver surgery. While inflammation contributes to the damage resulting from IRI, only a limited number of inflammation biomarkers have been identified as being associated with the different stages of hepatic IRI. As an approach to identify some of these inflammatory cytokines and the molecular mechanisms involved within different stages of hepatic IRI, we used an advanced antibody array assay to detect multiple proteins. With this technology, we observed specific differences in the content of inflammatory cytokines between ischemic and sham controls, as well as a function of the different reperfusion stages in a hepatic IRI mouse model. For example, while liver tissue content of IL-12p40/p70 was significantly increased in the ischemic stage, it was significantly decreased in the reperfusion stage as compared with that of the sham group. For other inflammatory cytokines, no changes were obtained between the ischemic and reperfusion stages with levels of IL-17, Eotaxin-2, Eotaxin, and sTNF RII all being consistently increased, while those of TIMP-1, TIMP-2, BLC, and MCSF consistently decreased as compared with that of the sham group at all reperfusion stages examined. Results from protein function annotation Gene Ontology and the KEGG pathway revealed that inflammatory cytokines are enriched in a network associated with activation of inflammatory response signaling pathways such as TLR, TNF, and IL-17 when comparing responses of the IR versus sham groups. The identification of cytokines along with their roles at specific stages of IRI may reveal important new biological markers for the diagnosis and prognosis of hepatic IRI.

Role of reactive oxygen species at reperfusion stage in isoflurane preconditioning-induced neuroprotection

In this in vivo and in vitro study, we aimed to investigate whether isoflurane preconditioning-induced neuronal protection is mediated by reactive oxygen species (ROS) signaling at the reperfusion stage. In the in vivo study, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) and in the in vitro study, rat pheochromocytoma (PC12) cells were subjected to oxygen glucose deprivation (OGD). Isoflurane preconditioning was carried out prior to MCAO or OGD and the ROS scavenger, N-2-mercaptopropiopylglycine (2-MPG), was administered at the start of reperfusion. Infarct volume, neurological severity score, and TUNEL staining were analyzed in the in vivo study and cell viability, Bcl-2/Bax ratio, cleaved caspase 3/caspase 3 ratio, and ROS fluorescence intensity were measured in the in vitro study. In the in vivo study, infarct volume, neurological severity score, and TUNEL-positive cell count were significantly decreased with preconditioning but were abrogated by administration of 2-MPG. In the in vitro study, cell viability and Bcl-2/Bax ratio were significantly increased with preconditioning, and cleaved caspase-3/caspase-3 ratio and ROS fluorescence intensity were significantly decreased. Administration of 2-MPG for 10 min abrogated this preconditioning effect, but it did not abolish the protection when administered for 60 min of reperfusion. Isoflurane preconditioning-induced protection was abolished by ROS scavengers at the start of reperfusion, indicating that ROS signaling can mediate the isoflurane preconditioning effect, which suggests that the time window can be important.

Measurement of Interhemispheric Correlation Coefficient in Rodent Model of Middle Cerebral Artery Occlusion Using Near Infrared Spectroscopy.

Near-infrared spectroscopy (NIRS) is a noninvasive brain imaging technique that measures hemodynamics by determining the optical properties of tissue. Clinical potential of NIRS for monitoring cerebral hemodynamics in cerebrovascular diseases, such as stroke, has been studied. However, inconsistencies in measurements among studies, which are believed to be partly due to anatomical variance and diversity in disease presentation, limit the clinical feasibility of NIRS for stroke monitoring. In the present study, bihemispheric frequency-domain NIRS measurements on middle cerebral artery occlusion rats were performed. The discrepancy in interhemispheric synchronicity in hemodynamic oscillation appeared during the early reperfusion stage is related to the size of infarct that developed three days later. These NIRS parameters may have the potential to be early prognostic biomarkers for long-term stroke monitoring in the future translational investigation.

Front Cover: In vivo label‐free functional photoacoustic monitoring of ischemic reperfusion (J. Biophotonics 7/2019)

How does the ischemic tissue re‐vascularize? Now we can visualize the reperfusion process at high spatial resolution by using a dual‐wavelength MEMS scanning based optical resolution photoacoustic microscopy (OR‐PAM) system. The fast imaging capability enables continuous monitoring of skin reperfusion in a mouse model. It's also found that the ischemic tissue has a significantly higher oxygen consumption rate in the reperfusion stage comparing to the normal tissue.Further details can be found in the article by Renzhe Bi, U.S. Dinish, Chi Ching Goh, et al. (e201800454). image

S100a8/a9 Signaling Causes Mitochondrial Dysfunction and Cardiomyocyte Death in Response to Ischemic/Reperfusion Injury.

Myocardial ischemia-reperfusion (MI/R) injury is a significant clinical problem without effective therapy. Unbiased omics approaches may reveal key MI/R mediators to initiate MI/R injury. We used a dynamic transcriptome analysis of mouse heart exposed to various MI/R periods to identify S100a8/a9 as an early mediator. Using loss/gain-of-function approaches to understand the role of S100a8/a9 in MI/R injury, we explored the mechanisms through transcriptome and functional experiment. Dynamic serum S100a8/a9 levels were measured in patients with acute myocardial infarction before and after percutaneous coronary intervention. Patients were prospectively followed for the occurrence of major adverse cardiovascular events. S100a8/a9 was identified as the most significantly upregulated gene during the early reperfusion stage. Knockout of S100a9 markedly decreased cardiomyocyte death and improved heart function, whereas hematopoietic overexpression of S100a9 exacerbated MI/R injury. Transcriptome/functional studies revealed that S100a8/a9 caused mitochondrial respiratory dysfunction in cardiomyocytes. Mechanistically, S100a8/a9 downregulated NDUF gene expression with subsequent mitochondrial complex I inhibition via Toll-like receptor 4/Erk-mediated Pparg coactivator 1 alpha/nuclear respiratory factor 1 signaling suppression. Administration of S100a9 neutralizing antibody significantly reduced MI/R injury and improved cardiac function. Finally, we demonstrated that serum S100a8/a9 levels were significantly increased 1 day after percutaneous coronary intervention in patients with acute myocardial infarction, and elevated S100a8/a9 levels were associated with the incidence of major adverse cardiovascular events. Our study identified S100a8/a9 as a master regulator causing cardiomyocyte death in the early stage of MI/R injury via the suppression of mitochondrial function. Targeting S100a8/a9-intiated signaling may represent a novel therapeutic intervention against MI/R injury. URL: https://www.clinicaltrials.gov. Unique identifier: NCT03752515.

Heat Shock Protein 70 Messenger RNA in Rat Leukocytes Elevates After Severe Intestinal Ischemia–Reperfusion

BackgroundHeat shock protein 70 (HSP70) confers protection against heat shock, oxidative stress, infection, and inflammation in many cell types. A recent study reported that the induction of HSP70 was associated with morphologic protection against ischemia–reperfusion injury (IRI) in the rat small intestine. This study investigated the dynamics of HSP70 in leukocytes during intestinal IRI in a rat model. Materials and methodsSerial blood samples were collected at 60-minute intervals up to 240 min from male Wistar rats (n = 15). The rats were divided into three groups of five each: the control group, the nonlethal IRI group, and the lethal IRI group. Rats belonging to the control group underwent a sham operation, and laparotomy was performed on rats in the lethal and nonlethal IRI groups. The nonlethal group experienced a 30-minute clamping of the superior mesenteric artery, and the lethal group experienced a 75-minute clamping of the superior mesenteric artery. The expression of HSP70 messenger RNA (mRNA) in leukocytes was measured by real-time quantitative polymerase chain reaction. Mixed-effects modeling of repeated measures was used to carry out the statistical analysis. The Bonferroni correction was applied to multiple comparisons. A P value < 0.0167 was considered to indicate statistical significance. ResultsThe expression of HSP70 mRNA in leukocytes increased 60 min after reperfusion in both IRI groups, and it was 12.8 times higher in the lethal group and 3.6 times higher in the nonlethal group compared with the control group. The expression of mRNA in the lethal group was significantly increased compared with the nonlethal group and the control group at 120 and 180 min after reperfusion. At 120 min after reperfusion, the expression of HSP70 mRNA was 6.1 times higher in the lethal group than in the nonlethal group (P = 0.0075) and 17.7 times higher than in the control group (P = 0.0011). At 180 min after reperfusion, the expression of HSP70 mRNA was 6.8 times higher in the lethal group than in the nonlethal group (P = 0.0007) and 4.3 times higher than in the control group (P = 0.0032). Although the expression of HSP70 mRNA in the nonlethal group was elevated in the early stages of reperfusion, there was no difference between the nonlethal group and the control group (P = 0.0212 at 60 min). ConclusionsThe expression of HSP70 mRNA in leukocytes may be a clinically useful indicator for evaluating pathologic conditions in intestinal IRI.

Global brain ischemia in rats is associated with mitochondrial release and downregulation of Mfn2 in the cerebral cortex, but not the hippocampus.

Mitochondria are crucial for neuronal cell survival and death through their functions in ATP production and the intrinsic pathway of apoptosis. Mitochondrial dysfunction is considered to play a central role in several serious human diseases, including neurodegenerative diseases, such as Parkinson's and Alzheimer's disease and ischemic neurodegeneration. The aim of the present study was to investigate the impact of transient global brain ischemia on the expression of selected proteins involved in mitochondrial dynamics and mitochondria-associated membranes. The main foci of interest were the proteins mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), voltage-dependent anion-selective channel 1 (VDAC1) and glucose-regulated protein 75 (GRP75). Western blot analysis of total cell extracts and mitochondria isolated from either the cerebral cortex or hippocampus of experimental animals was performed. In addition, Mfn2 was localized intracellularly by laser scanning confocal microscopy. It was demonstrated that 15-min ischemia, or 15-min ischemia followed by 1, 3, 24 or 72 h of reperfusion, was associated with a marked decrease of the Mfn2 protein in mitochondria isolated from the cerebral cortex, but not in hippocampal mitochondria. Moreover, a translocation of the Mfn2 protein to the cytoplasm was documented immediately after global brain ischemia in the neurons of the cerebral cortex by laser scanning confocal microscopy. Mfn2 translocation was followed by decreased expression of Mfn2 during reperfusion. Markedly elevated levels of the VDAC1 protein were also documented in total cell extracts isolated from the hippocampus of rats after 15 min of global brain ischemia followed by 3 h of reperfusion, and from the cerebral cortex of rats after 15 min of global brain ischemia followed by 72 h of reperfusion. The mitochondrial Mfn2 release observed during the early stages of reperfusion may thus represent an important mechanism of mitochondrial dysfunction associated with neuronal dysfunction or death induced by global brain ischemia.

Inhibition of Calcium/Calmodulin-Dependent Protein Kinase IIα Suppresses Oxidative Stress in Cerebral Ischemic Rats Through Targeting Glucose 6-Phosphate Dehydrogenase

Ischemic stroke is a leading cause of mortality and morbidity worldwide, and oxidative stress plays a significant role in the ischemia stage and reperfusion stage. Previous studies have indicated that both calcium/calmodulin-dependent protein kinase II (CaMKII) and glucose 6-phosphate dehydrogenase (G6PD) are involved in the oxidative stress. Thus, the aim of this study was to investigate the roles of CaMKIIα, an important isoform of CaMKII, and G6PD in a rat model of middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of small interfering ribonucleic acid (siRNA) for CaMKIIα was performed at 48h pre-MCAO surgery. Immunofluorescence Staining and western blot were performed to detect the expression of p-CaMKIIα and G6PD in the cortices. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was performed to investigate the infarct volume. In addition, neurological deficit, reactive oxygen species (ROS), ratio of reduced-to-oxidized glutathione (GSH/GSSG) and ratio of reduced-to-oxidized oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP+) were assessed. The results indicated that both p-CaMKIIα and G6PD were widely located in the neurons and astrocytes, and their expression was gradually increased in the cortices after MCAO, which was accompanied by increased level of ROS and decreased levels of GSH/GSSG and NADPH/NADP+. However, after treatment with siRNA for CaMKIIα, p-CaMKIIα expression was decreased and G6PD expression was increased. Moreover, inhibition of CaMKIIα improved the neurological deficit, reduced the infarct volume, decreased the level of ROS and increased the levels of GSH/GSSG and NADPH/NADP+. The results suggested that CaMKIIα inhibition exerted neuroprotectiveeffects through regulating G6PD expression, which provides a new target for prevention and treatment of stroke.

MicroRNA-124 expression in the brains of rats during early cerebral ischemia and reperfusion injury is associated with cell apoptosis involving STAT3.

Cerebral ischemia and reperfusion injury is a cause of death and disability in adults. MicroRNA-124 possesses protective effects against apoptosis in cerebral ischemia and reperfusion. To provide insights into the diagnosis and treatment of cerebral ischemia and reperfusion injury, the dynamic changes of microRNA-124 expression during the early stage of cerebral ischemia and reperfusion injury in rats was investigated by quantitative polymerase chain reaction. To elucidate the association between the dynamic expression of microRNA-124 and apoptosis, the expression of proteins associated with apoptosis, including caspase-3, apoptosis regulator Bcl-2 (Bcl-2) and apoptosis regulator Bax (Bax) was analyzed by immunohistochemistry and western blot analyses. As signal transducer and activator of transcription 3 (STAT3) is involved in cell apoptosis and associated with Bcl-2, the protein expression of STAT3 and its active form, phosphorylated (p-)STAT3, were analyzed by western blot analysis. The expression of microRNA-124 increased and the maximum value appeared 12 h after reperfusion. Similarly, the expression of Bcl-2 also peaked 12 h after reperfusion, however the expression of caspase-3 and Bax continued to increase after the 12 h time point. These results indicate that the expression of microRNA-124 is closely associated with Bcl-2 and serves a protective role, inhibiting apoptosis. As the upstream regulator of Bcl-2, the expression of p-STAT3 was in accordance with Bcl-2 expression and peaked 12 h after reperfusion. By contrast, STAT3 was downregulated and the minimum level of STAT3 protein was reached 12 h after reperfusion. In summary, during the early stage of cerebral ischemia and reperfusion, the dynamic expression of microRNA-124 exhibited protective effects through the inhibition of apoptosis via anti-apoptotic proteins Bcl-2 and STAT3. Conversely, caspase-3 and Bax maintain apoptosis. The present study provides evidence to aid in the understanding of cerebral ischemia and reperfusion injury and develops methods of diagnosis and therapy of this condition.

The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A.

Reactive oxygen species (ROS) are by-products of physiological mitochondrial metabolism that are involved in several cellular signaling pathways as well as tissue injury and pathophysiological processes, including brain ischemia/reperfusion injury. The mitochondrial respiratory chain is considered a major source of ROS; however, there is little agreement on how ROS release depends on oxygen concentration. The rate of H2 O2 release by intact brain mitochondria was measured with an Amplex UltraRed assay using a high-resolution respirometer (Oroboros) equipped with a fluorescent optical module and a system of controlled gas flow for varying the oxygen concentration. Three types of substrates were used: malate and pyruvate, succinate and glutamate, succinate alone or glycerol 3-phosphate. For the first time we determined that, with any substrate used in the absence of inhibitors, H2 O2 release by respiring brain mitochondria is linearly dependent on the oxygen concentration. We found that the highest rate of H2 O2 release occurs in conditions of reverse electron transfer when mitochondria oxidize succinate or glycerol 3-phosphate. H2 O2 production by complex III is significant only in the presence of antimycin A and, in this case, the oxygen dependence manifested mixed (linear and hyperbolic) kinetics. We also demonstrated that complex II in brain mitochondria could contribute to ROS generation even in the absence of its substrate succinate when the quinone pool is reduced by glycerol 3-phosphate. Our results underscore the critical importance of reverse electron transfer in the brain, where a significant amount of succinate can be accumulated during ischemia providing a backflow of electrons to complex I at the early stages of reperfusion. Our study also demonstrates that ROS generation in brain mitochondria is lower under hypoxic conditions than in normoxia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

BMSCs protect against renal ischemia-reperfusion injury by secreting exosomes loaded with miR-199a-5p that target BIP to inhibit endoplasmic reticulum stress at the very early reperfusion stages.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been recently reported to play a variety of vital roles in organ and tissue damage repair, mainly via potent paracrine activity, including secreting extracellular vesicles, such as exosomes, that serve as mediators facilitating intercellular communication and reprogramming recipient cells by delivering their contents to target cells. However, the underlying mechanisms are diverse and complex, and the influencing characteristics have rarely been studied. Accordingly, we designed this study to explore the time dependence of the effects of exosomes derived from BMSCs (BMexos) on renal ischemia-reperfusion (I/R) injury and the underlying mechanisms associated with the reperfusion time. Impressively, our study is the first to find that BMexos protected against renal I/R injury in vitro and in vivo at the very early reperfusion stages, especially 4-8 h after reperfusion in vitro and 8-16 h after reperfusion in vivo. Interestingly, we simultaneously found that endoplasmic reticulum (ER) stress was significantly suppressed following the administration of BMexos in vitro and in vivo with a similar time dependence. Additionally, we discovered that miR-199a-5p, which was abundant in the BMSCs, was transferred into renal tubular epithelial cells (NRK-52E) in a time-dependent manner and significantly inhibited I/R-induced ER stress by targeting binding immunoglobulin protein (BIP). Cocultivation with miR-199a-5p-overexpressing BMSCs amplified the suppression of ER stress and further protected against I/R injury. However, coculture with miR-199a-5p-knockdown BMSCs obviously increased ER stress and reversed the BMexos-induced protection, and silencing BIP by small interfering RNA-1098 in NRK-52E inhibited these effects. This study provides evidence that administering BMexos at the very early reperfusion stages significantly protects against renal I/R injury, and ER stress is closely linked to this protection. These results suggest a novel therapeutic strategy during the very early reperfusion stages of renal I/R injury.-Wang, C., Zhu, G., He, W., Yin, H., Lin, F., Gou, X., Li, X. BMSCs protect against renal ischemia-reperfusion injury by secreting exosomes loaded with miR-199a-5p that target BIP to inhibit endoplasmic reticulum stress at the very early reperfusion stages.

MTOR and Beclin1: Two key autophagy-related molecules and their roles in myocardial ischemia/reperfusion injury.

Autophagy is the general term of lysosomal degradation of substances in cells, which is considered the key to maintaining the normal structure and function of the heart. It also has a correlation with several heart diseases, in particular, myocardial ischemia/reperfusion (I/R) injury. At the stage of myocardial ischemia, autophagy degrades nonfunctional cytoplasmic proteins providing the critical nutrients for the critical life activities, thereby suppressing cell apoptosis and necrosis. However, autophagy is likely to affect the heart negatively in the reperfusion stage. Mammalian target of rapamycin (mTOR) and Beclin1 are two vital autophagy-related molecules in myocardial I/R injury playing significant roles in different stages. In the ischemia stage, mTOR plays its roles through AMPK/mTOR and phosphoinositide 3-kinase/Akt/mTOR pathway, whereas Beclin1 plays its roles through its upregulation in the reperfusion stage. A possible interaction between mTOR and Beclin1 has been reported recently, and further studies need to be done to find the underlying interaction between the two molecules in myocardial I/R injury.

Effects of melatonin on acute brain reperfusion stress: role of Hippo signaling pathway and MFN2-related mitochondrial protection.

Acute brain reperfusion stress is associated with mitochondrial dysfunction through unknown mechanisms. Accordingly, there is no effective drug to control the development and progression of brain reperfusion stress currently. The aim of our investigation is to verify whether melatonin attenuates acute brain reperfusion stress via affecting mitochondrial function. Our studies demonstrated that melatonin treatment suppressed reperfusion-induced neuron death. At the molecular levels, melatonin treatment modulated mitochondrial homeostasis via activating mitochondrial fusion. At the stage of reperfusion, MFN2 expression was downregulated, contributing to mitochondrial fusion inhibition. Interestingly, MFN2-related mitochondrial fusion was reversed by melatonin. Loss of MFN2-related mitochondrial fusion abrogated the protective actions of melatonin on mitochondrial function. Mechanistically, melatonin sustained MFN2-related mitochondrial fusion via suppressing Mst1-Hippo pathway. Overexpression of Mst1 attenuated the beneficial effects of melatonin on mitochondrial fusion, evoking mitochondrial damage and neuron death in the setting of brain reperfusion stress. Taken together, our results confirmed the protective effects of melatonin on acute brain reperfusion stress. Melatonin treatment activated MFN2-related mitochondrial fusion via suppressing Mst1-Hippo pathway, finally sustaining mitochondrial function and reducing reperfusion-mediated cerebral injury.

Hypoxic preconditioning attenuates necroptotic neuronal death induced by global cerebral ischemia via Drp1-dependent signaling pathway mediated by CaMKIIα inactivation in adult rats.

Hypoxic preconditioning (HPC) alleviates the selective and delayed neuronal death in the hippocampal CA1 region induced by transient global cerebral ischemia (tGCI). This type of cell death may include different programmed cell death mechanisms, namely, apoptosis and necroptosis. Although apoptotic signaling is well defined, the mechanisms that underlie neuronal necroptosis are yet to be fully elucidated. In this study, we investigated whether HPC protects neurons from cerebral ischemia-induced necroptosis. We observed that tGCI up-regulated the expression of receptor-interacting protein (RIP) 3 and increased the interaction of RIP1-RIP3 in CA1 at the early stage of reperfusion. The pretreatment with HPC or necrostatin-1 decreased the expression of RIP3 and the formation of RIP1-RIP3 after tGCI. We also found that HPC decreased the expression and the activity of caspase-8 in CA1 after tGCI, and notably, the pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, did not trigger necroptosis but attenuated the tGCI-induced neuronal damage. Furthermore, we demonstrated that HPC decreased the activation of calcium-calmodulin kinase (CaMK) IIα and the interaction of RIP1 and CaMKIIα induced by tGCI. Intriguingly, the pretreatment with a CaMKs inhibitor KN-93 before tGCI resulted in significantly reduced RIP1-3 interaction and tGCI-induced neuronal damage. Finally, we ascertained that HPC prevented the dephosphorylation of dynamin-related protein 1 (Drp1)-Ser637 (serine 637) and inhibited the translocation of Drp1 to mitochondria induced by tGCI. Importantly, the treatment with a Drp1 inhibitor Mdivi-1 or necrostatin-1 before tGCI also abolished Drp1 dephosphorylation at Ser637 and mitochondrial translocation. Taken together, our results highlight that HPC attenuates necroptotic neuronal death induced by tGCI via Drp1-dependent mitochondrial signaling pathways mediated by CaMKIIα inactivation.-Zhan, L., Lu, Z., Zhu, X., Xu, W., Li, L., Li, X., Chen, S., Sun, W., Xu, E. Hypoxic preconditioning attenuates necroptotic neuronal death induced by global cerebral ischemia via Drp1-dependent signaling pathway mediated by CaMKIIα inactivation in adult rats.