Early Stage Of Reperfusion Research Articles

A Membrane-Targeting Aggregation-Induced Emission Probe for Monitoring Lipid Droplet Dynamics in Ischemia/Reperfusion-Induced Cardiomyocyte Ferroptosis.

Myocardial ischemia/reperfusion injury (MIRI) is the leading cause of irreversible myocardial damage. A pivotal pathogenic factor is ischemia/reperfusion (I/R)-induced cardiomyocyte ferroptosis, marked by iron overload and lipid peroxidation. However, the impact of lipid droplet (LD) changes on I/R-induced cardiomyocyte ferroptosis is unclear. In this study, an aggregation-induced emission probe, TPABTBP is developed that is used for imaging dynamic changes in LD during myocardial I/R-induced ferroptosis. TPABTBP exhibits excellent LD-specificity, superior capability for monitoring lipophagy, and remarkable photostability. Molecular dynamics (MD) simulation and super-resolution fluorescence imaging demonstrate that the TPABTBP is specifically localized to the phospholipid monolayer membrane of LDs. Imaging LDs in cardiomyocytes and myocardial tissue in model mice with MIRI reveals that the LD accumulation level increase in the early reperfusion stage (0-9h) but decrease in the late reperfusion stage (>24h) via lipophagy. The inhibition of LD breakdown significantly reduces the lipid peroxidation level in cardiomyocytes. Furthermore, it is demonstrated that chloroquine (CQ), an FDA-approved autophagy modulator, can inhibit ferroptosis, thereby attenuating MIRI in mice. This study describes the dynamic changes in LD during myocardial ischemia injury and suggests a potential therapeutic target for early MIRI intervention.

Iron promotes both ferroptosis and necroptosis in the early stage of reperfusion in ischemic stroke

Programmed cell death contributes to neurological damage in ischemic stroke, especially during the reperfusion stage. Several cell death pathways have been tested preclinically and clinically, including ferroptosis, necroptosis, and apoptosis. However, the sequence and complex interplay between cell death pathways during ischemia/reperfusion remains under investigation. Here, we unbiasedly investigated cell death pathways during ischemia/reperfusion by utilizing RNA sequencing analysis and immunoblot assays and revealed that ferroptosis and necroptosis occurred early post-reperfusion, followed by apoptosis. Ferroptosis inhibitor Liproxstatin-1 effectively inhibited necroptosis during reperfusion, while the necroptosis inhibitor Necrostatin-1 suppressed protein expression consistent with ferroptosis activation. Protein–protein interaction analysis and iron chelation therapy by deferoxamine mesylate indicate that iron is capable of promoting both ferroptosis and necroptosis in middle cerebral artery occlusion/repression modeled mice. Treatment of cells with iron led to a disruption in redox balance with activated necroptosis and increased susceptibility to ferroptosis. Collectively, these data uncovered a complex interplay between ferroptosis and necroptosis during ischemic stroke and indicated that multiple programmed cell death pathways may be targeted co-currently.

Alpha-lipoic acid enhances ischemic postconditioning-mediated improvement of myocardial infarction and apoptosis in diabetic rats with ischemia/reperfusion injury.

This work evaluated the combined effects of alpha-lipoic acid (ALA) and ischemic postconditioning (Post) on myocardial infarction and cell death in rats with chronic type-II diabetes following ischemia/reperfusion injury. The rats received a high-fat diet and were given one intraperitoneal injection of 35 mg/kg streptozotocin to induce chronic diabetes. They were then pretreated with ALA (100 mg/kg/day, orally) for 5 weeks before undergoing ischemia/reperfusion (I/R) insult. The hearts experienced 35 min regional ischemia through ligating the left anterior descending coronary artery, followed by 60 min reperfusion. The Post protocol involved 6 cycles of a 10/10 s algorithm, applied during the early stage of reperfusion. The use of Post alone did not significantly alter lactate dehydrogenase and infarct size levels, while ALA showed positive effects. Similar findings were observed for apoptotic changes with single treatments. However, the concurrent administration of ALA and Post significantly reduced the protein expressions of Bax, Bax/Bcl2, and cleaved caspase-3 while increasing Bcl2 expression. Additionally, the histopathological findings of the combined therapy were superior to those of single treatments. The concomitant use of ALA and Post effectively inhibited apoptosis, leading to cardiac recovery after I/R injury in diabetic conditions. This strategy could improve outcomes for preserving diabetic hearts following I/R insults.

Ischemic-Postconditioning Improves Myocardial Injury and Fibrosis Following Ischemia/Reperfusion Injury in Diabetic Rats Pretreated with Alpha-Lipoic Acid

Background: Myocardial ischemia/reperfusion (I/R) injury is one of the pre-eminent causes of death. Prevention of I/R injury in diabetic patients is full of ups and downs. This study has investigated the effect of co-administration of alpha-lipoic acid (ALA) preconditioning and ischemic-postconditioning (Post) on myocardial injury and fibrosis in type-II diabetic rats suffering from I/R injury. Methods: Chronic diabetes was induced by a high-fat supplemented diet and low-dose streptozotocin within 3 months. Diabetic rats received ALA (100 mg/kg/d, orally) for 5 weeks before I/R induction. The myocardium underwent 35 minutes of ischemia through left anterior descending (LAD) coronary artery ligation and 60 minutes of reperfusion. Post was consisted of six cycles of 10/10 seconds algorithm and applied in the early stage of reperfusion. The release of creatine kinase (CK) as the injury enzyme was measured with ELISA. The protein expression of fibrosis markers was evaluated with western blotting and tissue fibrosis was scored through Masson’s trichrome staining. Results: Post alone had no considerable effect on cardiac injury marker CK, while ALA had somehow a positive impact. However, the application of Post in ALA-pretreated rats significantly reduced the injury marker in comparison with the untreated I/R group. In addition, this combination therapy decreased Smad3 and transforming growth factor-β (TGF-β) expression and improved tissue fibrosis as compared with the untreated group. Conclusion: Combination of ALA pretreatment and Post evoked a cardio-protective effect by improving myocardial injury and attenuating fibrosis induced by diabetes. This combined treatment can be recommended as a useful strategy to restore cardio-protection in hearts with diabetic comorbidity.

The neuroprotective role of prolonged normobaric oxygenation applied during ischemia and in the early stage of reperfusion in cerebral ischemic rats

BackgroundRecanalization is the main treatment option for ischemic stroke. However, prognosis remains poor for about half of patients after recanalization, possibly due to the “no-reflow” phenomenon at the early phase of recanalization. Normobaric oxygenation (NBO) during ischemia can reportedly maintain the partial pressure of oxygen and exert a protective effect in ischemic brain tissue. Objectives and methodsThis study investigated whether prolonged NBO treatment during ischemia and the early phase of reperfusion (i/rNBO) has neuroprotective effects and to elucidate the underlying mechanisms in rats with middle cerebral artery occlusion plus reperfusion. ResultsNBO treatment significantly elevated the level of O2 in the atmosphere and arterial blood without altering the level of CO2. The infarcted cerebral volume was significantly reduced by application of i/rNBO as compared to iNBO (applied during ischemia) or rNBO (applied at the early phase of reperfusion), indicating better protective effects of i/rNBO. i/rNBO more effectively suppressed s-nitrosylation of MMP-2 (amplifying inflammation) as compared to iNBO and rNBO, dramatically downregulated the cleavage of poly(ADP-ribose)polymerase-1 (PARP-1, acting as the substrate of MMP-2), and suppressed neuronal apoptosis, as determined by the TUNEL assay and staining for NeuN. These results demonstrated that application of i/rNBO in the early stage of reperfusion significantly alleviated neuronal apoptosis via suppression of the MMP-2/PARP-1 pathway. ConclusionsThe mechanism underlying the neuroprotective role of i/rNBO involved prolonged NBO treatment for cerebral ischemia, suggesting that i/rNBO may allow expansion of the time window for NBO application in stroke patients following vascular recanalization.

S100-A8/A9 activated TLR4 in renal tubular cells to promote ischemia-reperfusion injury and fibrosis.

Renal ischemia/reperfusion injury (IRI) is a significant clinical problem without effective therapy. Unbiased omics approaches may reveal key renal mediators to initiate IRI. S100-A8/A9 was identified as the most significantly upregulated gene and protein base on proteomic analysis and RNA sequencing during the early reperfusion stage. S100-A8/A9 levels were significantly increased 1day after transplantation in patients with donation after brain death (DBD). S100-A8/A9 production was associated with CD11b+Ly6G+ CXCR2+ immunocytes infiltration. Administration of S100-A8/A9 blocker ABR238901 significantly alleviates renal tubular injury, inflammatory cell infiltration, and renal fibrosis after renal IRI. Mechanistically, S100-A8/A9 could promote renal tubular cell injury and profibrotic cytokine production via TLR4. In conclusion, our findings found that early activation of S100-A8/A9 in renal IRI and targeting S100-A8/A9 signaling alleviates tubular injury and inhibits inflammatory response and renal fibrosis, which may provide a novel target for the prevention and treatment of acute kidney injury.

Characterization of the Involvement of Tumour Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6) in Ischemic Brain Injury Caused by Middle Cerebral Artery Occlusion in Mouse.

The identification of novel targets to modulate the immune response triggered by cerebral ischemia is crucial to promote the development of effective stroke therapeutics. Since tumour necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, is involved in the regulation of immune and stromal cell functions in acute neurodegeneration, we aimed to characterize its involvement in ischemic stroke. Transient middle cerebral artery occlusion (1 h MCAo, followed by 6 to 48 of reperfusion) in mice resulted in a significant elevation in cerebral TSG-6 protein levels, mainly localized in neurons and myeloid cells of the lesioned hemisphere. These myeloid cells were clearly infiltrating from the blood, strongly suggesting that brain ischemia also affects TSG-6 in the periphery. Accordingly, TSG-6 mRNA expression was elevated in peripheral blood mononuclear cells (PBMCs) from patients 48 h after ischemic stroke onset, and TSG-6 protein expression was higher in the plasma of mice subjected to 1 h MCAo followed by 48 h of reperfusion. Surprisingly, plasma TSG-6 levels were reduced in the acute phase (i.e., within 24 h of reperfusion) when compared to sham-operated mice, supporting the hypothesis of a detrimental role of TSG-6 in the early reperfusion stage. Accordingly, systemic acute administration of recombinant mouse TSG-6 increased brain levels of the M2 marker Ym1, providing a significant reduction in the brain infarct volume and general neurological deficits in mice subjected to transient MCAo. These findings suggest a pivotal role of TSG-6 in ischemic stroke pathobiology and underscore the clinical relevance of further investigating the mechanisms underlying its immunoregulatory role.

Effects of Endothelin-1 and nitric oxide levels on myocardial ischemia-reperfusion injury.

To study the role of nitric oxide (NO) in myocardial ischemia-reperfusion injury (IRI) and Endothelin-1 (ET-1) in the process of reperfusion in an animal model. ET is a strong vasoconstrictor peptide, which is closely related to the physiological and pathological state of the cardiovascular system. ET not only directly stimulates and activates a variety of hormones and cytokines, but also is one of the mediators promoting myocardial remodeling, and participates in and promotes myocardial ischemia injury. Before myocardial ischemia, Krebs-Henseleit (KH) perfusion solution containing different concentrations of L-arginine (LA; substrate of NO) were given to 6 groups of rats, and ET was given at the early stage of reperfusion in 3 groups. During reperfusion, cardiac function indexes, myocardial enzyme release and NO content in coronary effluent, and the cardiac malondialdehyde (MDA) content was measured. The myocardial ultrastructure was observed by microscopy. Data of each group are expressed as mean ± standard deviation, and the baseline value of each group before ischemia was the recovery value during reperfusion. SPSS26.0 (IBM, Chicago, USA) was used for statistical processing. Before myocardial ischemia, infusion of KH solution containing a low concentration of LA (10 mmol/L) reduced myocardial IRI, whereas infusion of a KH solution containing high concentration of LA (100 mmol/L) before ischemia significantly aggravated myocardial IRI. The administration of KH solution containing LA and ET-1 (1,000 mmol/L) significantly reduced myocardial IRI. NO plays a dual role in myocardial ischemia-reperfusion, both beneficial and harmful. The combination of NO and ET-1 can reduce the toxic effect of NO.

A novel function of ATF3 in suppression of ferroptosis in mouse heart suffered ischemia/reperfusion

IntroductionFerroptosis, a newly identified type of programmed cell death type, has been proven to contribute to the progression of myocardial ischemia/reperfusion (I/R) injury. However, little is known about ferroptosis regulation in I/R injury. ObjectivesWe identified activating transcription factor 3 (ATF3) as a vital regulator of I/R induced ferroptosis and investigated the effects and potential mechanism of ATF3 in cardiac ferroptosis. MethodsIn this study, the dynamic RNA-sequencing (RNA-seq) analysis were performed on mouse hearts exposed to different I/R schedules to identify that ATF3 represents an important modulatory molecule in myocardial I/R injury. Then knockout, rescue and overexpression methods were used in mice and neonatal mouse cells (NMCs) to illustrate the effect of ATF3 on myocardial I/R injury. Loss/gain of function techniques were used both in vivo and in vitro to explore the effects of ATF3 on ferroptosis in I/R injury. Furthermore, chromatin immunoprecipitation sequence (ChIP-seq) analysis was performed in the AC16 human cardiomyocyte cell line to investigate potential genes regulated by ATF3. ResultsATF3 expression reached highest level at early stage of reperfusion, knockout of ATF3 significantly aggravated I/R injury, which could be rescued by ATF3 re-expression. Knockout and the re-expression of ATF3 changed the transcription levels of multiple ferroptosis genes. In addition, results showed that overexpression of ATF3 inhibits cardiomyocyte ferroptosis triggered by erastin and RSL3. Lastly, ChIP-seq and dual luciferase activity analysis revealed ATF3 could bind to the transcription start site of Fanconi anaemia complementation group D2 (FANCD2) and increased the FANCD2 promoter activity. Furthermore, we first demonstrated that overexpression of FANCD2 exerts significant anti-ferroptosis and cardioprotective effect on AC16 cell H/R injury. ConclusionATF3 inhibits cardiomyocyte ferroptotic death in I/R injury, which might be related with regulating FANCD2. Our study provides new insight into the molecular target for the therapy of myocardial I/R injury.

The zinc transporter ZIP7 (Slc39a7) controls myocardial reperfusion injury by regulating mitophagy.

Whereas elimination of damaged mitochondria by mitophagy is proposed to be cardioprotective, the regulation of mitophagy at reperfusion and the underlying mechanism remain elusive. Since mitochondrial Zn2+ may control mitophagy by regulating mitochondrial membrane potential (MMP), we hypothesized that the zinc transporter ZIP7 that controls Zn2+ levels within mitochondria would contribute to reperfusion injury by regulating mitophagy. Mouse hearts were subjected to ischemia/reperfusion in vivo. Mitophagy was evaluated by detecting mitoLC3II, mito-Keima, and mitoQC. ROS were measured with DHE and mitoB. Infarct size was measured with TTC staining. The cardiac-specific ZIP7 conditional knockout mice (ZIP7 cKO) were generated by adopting the CRISPR/Cas9 system. Human heart samples were obtained from donors and recipients of heart transplant surgeries. KO or cKO of ZIP7 increased mitophagy under physiological conditions. Mitophagy was not activated at the early stage of reperfusion in mouse hearts. ZIP7 is upregulated at reperfusion and ZIP7 cKO enhanced mitophagy upon reperfusion. cKO of ZIP7 led to mitochondrial depolarization by increasing mitochondrial Zn2+ and, accumulation of PINK1 and Parkin in mitochondria, suggesting that the decrease in mitochondrial Zn2+ in response to ZIP7 upregulation resulting in mitochondrial hyperpolarization may impede PINK1 and Parkin accumulation in mitochondria. Notably, ZIP7 is markedly upregulated in cardiac mitochondria from patients with heart failure (HF), whereas mitochondrial PINK1 accumulation and mitophagy were suppressed. Furthermore, ZIP7 cKO reduced mitochondrial ROS generation and myocardial infarction via a PINK1-dependet manner, whereas overexpression of ZIP7 exacerbated myocardial infarction. Our findings identify upregulation of ZIP7 leading to suppression of mitophagy as a critical feature of myocardial reperfusion injury. A timely suppression of cardiac ZIP7 upregulation or inactivation of ZIP7 is essential for the treatment of reperfusion injury.

Effect of Intravenous Lidocaine on Inflammatory and Apoptotic Response of Ischemia-Reperfusion Injury in Pigs Undergoing Lung Resection Surgery.

Background Ischemia-reperfusion injury is one of the most critical phenomena in lung transplantation and causes primary graft failure. Its pathophysiology remains incompletely understood, although the inflammatory response and apoptosis play key roles. Lidocaine has anti-inflammatory properties. The aim of this research is to evaluate the effect of intravenous lidocaine on the inflammatory and apoptotic responses in lung ischemia-reperfusion injury. Methods We studied the histological and immunohistochemical changes in an experimental model of lung transplantation in pigs. Twelve pigs underwent left pneumonectomy, cranial lobectomy, caudal lobe reimplantation, and 60 minutes of graft reperfusion. Six of the pigs made up the control group, while six other pigs received 1.5 mg/kg of intravenous lidocaine after induction and a 1.5 mg/kg/h intravenous lidocaine infusion during surgery. In addition, six more pigs underwent simulated surgery. Lung biopsies were collected from the left caudal lobe 60 minutes after reperfusion. We conducted a double study on these biopsies and assessed the degree of inflammation, predominant cell type (monocyte-macrophage, lymphocytes, or polymorphous), the degree of congestion, and tissue edema by hematoxylin and eosin stain. We also conducted an immunohistochemical analysis with antibodies against CD68 antigens, monocyte chemoattractant protein-1 (MCP-1), Bcl-2, and caspase-9. Results The lungs subjected to ischemia-reperfusion injury exhibited a higher degree of inflammatory infiltration. The predominant cell type was monocyte-macrophage cells. Both findings were mitigated by intravenous lidocaine administration. Immunohistochemical detection of anti-CD68 and anti-MCP-1 showed higher infiltration in the lungs subjected to ischemia-reperfusion injury, while intravenous lidocaine decreased the expression. Ischemia-reperfusion induced apoptotic changes and decreased Bcl-2 expression. The group treated with lidocaine showed an increased number of Bcl-2-positive cells. No differences were observed in caspase-9 expression. Conclusions In our animal model, intravenous lidocaine was associated with an attenuation of the histological markers of lung damage in the early stages of reperfusion.

USP30 protects against oxygen-glucose deprivation/reperfusion induced mitochondrial fragmentation and ubiquitination and degradation of MFN2.

Cerebral ischemia-reperfusion induces mitochondrial fragmentation and dysfunction, which plays a critical role in the subsequent neuronal death and neurological impairment. Protection of mitochondria is an effective strategy to prevent neuronal damage after cerebral ischemia-reperfusion injury. USP30 is a deubiquitinating enzyme that localizes to the outer mitochondrial membrane. USP30 participates in the regulation of mitophagy and maintenance of mitochondrial morphology. In this study, the neuroprotective effect of USP30 and the underlying mechanisms were assessed in an ischemia-reperfusion injury model. SK-N-BE (2) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. Ubiquitination of mitochondrial proteins is increased during the early stage of reperfusion after oxygen-glucose deprivation (OGD), but the ubiquitination of cytoplasmic proteins exhibits no obvious changes. OGDR insult also induces rapid ubiquitination and degradation of the mitochondrial fusion protein mitofusin 2 (MFN2) in the early stage of reperfusion after OGD. Overexpression of MFN2 attenuates OGDR induced mitochondrial fragmentation. USP30 overexpression suppresses OGDR-induced ubiquitination and degradation of MFN2, and protects against mitochondrial fragmentation. Therefore, precisely targeting USP30 may provide a novel therapeutic strategy for cerebral ischemia-reperfusion related disorders.

Characterization of novel lncRNAs in upper thoracic spinal cords of rats with myocardial ischemia-reperfusion injuries.

Myocardial ischemia-reperfusion injury (MIRI) is a significant problem in clinical cardiology, and refers to a more serious myocardial injury caused by blood recanalization after a period of myocardial ischemia, as compared with injury caused by vascular occlusion. The spinal cord, as the primary afferent and efferent center of cardiac sensory and sympathetic nerve fibres, has received increased attention in recent years with regards to the regulation of MIRIs. Previous studies have revealed that MIRI has a strong correlation with the abnormal expression of long non-coding (lnc)RNAs in the myocardium; however, there are limited reports on the effects of the altered expression of lncRNAs in the spinal cord following MIRI. To investigate the expression patterns of lncRNAs in the spinal cord after MIRI and their potential role in the early stage of reperfusion, a MIRI model was established in rats. After 30 min of myocardial ischemia and 2 h of reperfusion, the upper thoracic spinal cord tissues were immediately dissected and isolated. lncRNAs and mRNAs in spinal cord tissues were screened using transcriptome sequencing technology, and the expression of several highly deregulated mRNAs, including Frs3, Zfp52, Dnajc6, Nedd4l, Tep1, Myef2, Tgfbr1, Fgf12, Mef2c, Tfdp1 and lncRNA, including ENSRNOT00000080713, ENSRNOT00000090564, ENSRNOT00000082588, ENSRNOT00000091080, ENSRNOT00000091570, ENSRNOT00000087777, ENSRNOT00000082061, ENSRNOT00000091108, ENSRNOT00000087028, ENSRNOT00000086475, were further validated via reverse transcription-quantitative PCR. The number of altered expressed lncRNAs was 126, among which there were 41 upregulated probe sets and 85 downregulated sets. A total of 470 mRNAs were differentially expressed, in which 231 probe sets were upregulated and 239 were downregulated. Gene Ontology analysis indicated that dysregulated transcripts related to biological processes were mainly associated with ‘cell-cell signaling’. Moreover, pathway analysis demonstrated significant changes in the ‘PI3K/Akt signaling pathway’ and the ‘p53 signaling pathway’. Thus, the altered expression of lncRNAs in the spinal cord may be of considerable importance in the process of MIRI. The present results could provide an insight into the potential roles and mechanism of lncRNAs during the early stage of reperfusion.

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.

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.

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.

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/.