Minutes Of Reperfusion Research Articles

Apolipoprotein C3-rich low-density lipoprotein (AC3RL) increases BBB permeability and deteriorates cerebral ischemic stroke injury

Abstract Background Ischaemic stroke causes disability due to brain damage resulting from blood supply obstruction or increased permeability of the blood-brain barrier (BBB) that exposes the blood to toxic substances during reperfusion. Apolipoprotein C3 is a crucial triglyceride transport modulator, and its overexpression raises the risk of dyslipidemia and cardiovascular disease. Clinically, we observed that stroke patients have higher levels of apolipoprotein C3-rich low-density lipoprotein (AC3RL) expression than healthy individuals, and they also have worse prognoses. Simultaneously, we found that an increase in AC3RL is involved in endothelial cell apoptosis and inflammatory response, which may be linked to the occurrence of brain injury after stroke. Purpose This study aimed to explore whether the high expression of AC3RL would increase the permeability of the blood-brain barrier, and aggravate brain damage. Methods The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) experiment was carried out using C57BL/6 and apolipoprotein E gene knockout mice (ApoE-/-). WT receive an IV injection of AC3RL, which was separate from stroke patients' plasma. APOE-/- was fed a high-fat diet to promote AC3RL higher expression. We performed 60-minute ischaemic and 7-day reperfusion. The modified neurological severity score (mNSS) method was used to assess motor and neurological scores. Evans blue dye was injected through the tail vein one hour before euth (A) AC3RL, which was separated from stroke patients' plasma. (B) AC3RL concentration in individuals. (C) The white portion of the mice brains TTC stain indicates the site of the injury. (D) Evans blue dye leakage study in mice brains exhibited BBB permeability changes. (E) Assessment of mice's motor and neurological performance using the mNSS. anasia to assess BBB permeability. TTC staining of brain tissue after euthanasia to pinpoint the site of brain damage. Results Evans blue dye leakage was observed in the brain tissues of the group with high AC3RL expression, indicating that the BBB permeability has expanded. The mNSS method revealed a significant rise in mice with high levels of AC3RL expression, which suggested a decline in motor and nervous function. Conclusions Whether endogenous or exogenous, increased expression of AC3RL leads to more severe cerebral ischemic injury and motor dysfunction, which we believe may be due to increased BBB permeability caused by AC3RL. Therefore, AC3RL can be used as a disease predictor in the future, reducing AC3RL will be the main indicator for reducing severe brain injury.Figure 1.Figure 2.

Abstract 12179: Neutrophil-Specific Deletion of Beta2 Integrins Alleviates Myocardial Ischemia-Reperfusion Injury in Male Mice

Neutrophil recruitment and their mediated inflammatory responses are critical for cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury, which accounts for 9% mortality and 10% morbidity rates in ischemic heart disease patients. Blocking leukocyte recruitment in mouse knockouts (KO) of beta2 integrin (CD18) or blocking beta2 integrin with antibodies in multiple animals significantly reduced infarct size after myocardial I/R injury. However, the neutrophil-specific contribution of beta2 integrin to I/R injury is unknown. This study used the newly established CD18 flox/flox (hITGB2 KI) mouse strain to address this knowledge gap. We crossed them to MRP8-cre (S100A8) and confirmed the neutrophil-specific KO of beta2 integrins. Interestingly, all leukocyte subtypes were elevated in the blood of MRP8-cre CD18 flox/flox mice, indicating potential chronic inflammation. After 35 minutes of myocardial ischemia and 24 hours of reperfusion, we found MRP8-cre CD18 flox/flox mice have significantly reduced infarct size compared to cre- controls. However, if we distinguish the sex in analysis, we only saw a significant alleviation in male but not female MRP8-cre CD18 flox/flox mice. The quantification of leukocyte recruitment in hearts after I/R injury showed that male but not female MRP8-cre CD18 flox/flox mice have less recruitment of neutrophils and Ly6C high inflammatory monocytes. These results suggested sex bias of neutrophil-specific contributions of beta2 integrins in myocardial ischemia-reperfusion injury and provided new insights into beta2 integrin targeting therapies.

Abstract 13361: The Effect of Electronic Cigarette Exposure on Flow Mediated Vasodilation and LV Remodeling in a Chronic Myocardial Infarction Model

Introduction: There is little information about the effect of electronic cigarette (e-C) exposure on the cardiovascular system in the setting of myocardial infarction (MI) and whether e-C’s could alter vascular or cardiac function during the healing phase of MI. We determine the effect of e-C on endothelial function, cardiac remodeling and function during the healing phase of MI. Methods: Sprague Dawley rats (both sexes) were anesthetized and subjected to a proximal left coronary artery occlusion to induce a large anterior wall MI. At one week, rats were randomized to receive either 12 weeks of exposure to purified air (n=37) or e-cig vapor plus 15 mg/ml of nicotine (e-Cn=32). At 12 weeks endothelial function was tested by assessing femoral artery diameter and blood flow following a 5- minute femoral artery occlusion and reperfusion (ultrasound and flow probe). Cardiac function was assessed by echocardiography; remodeling was also assessed by quantitative histologic analysis. Results: Baseline blood flow in the femoral artery did not differ between groups, but peak reperfusion blood flow was blunted in the e-Cn group (1.59 ± 0.15 ml/min) vs. the air group (2.11 ± 0.18 ml/min; p=0.034). Femoral artery diameter after reperfusion was narrower in the e-Cn group (0.54 ± 0.02mm) compared to the air group (0.60 ± 0.02mm; p=0.023). Postmortem left ventricular (LV) volumes were similar in the e-Cn (0.69 ± 0.04 ml) and air groups (0.73 ± 0.04 ml; p=NS); and myocardial infarct expansion index did not differ between groups (1.4 ± 0.1 in e-Cn group versus 1.3 ± 0.1 in air group; p=NS). Gross pathology LV wall thickness was greater in the e-Cn group (1.38 ± 0.12 mm) versus the air group (1.04 ± 0.10 mm) despite no increase in blood pressure at 12 weeks in the e-CN group. LV fractional shortening by echo did not differ between groups at 12 weeks (e-Cn at 29 ± 2% and air at 27 ± 1%; p=NS). Conclusion: Exposure to e-Cn during the healing phase of MI was associated with altered endothelial function with reduced femoral artery blood flow and diameter at reperfusion, but not with worsened LV dilation or worsened cardiac function. E-Cn did increase gross pathologic LV wall thickness. E-Cn exposure during the healing phase of MI has deleterious effects on the CV system but does not dilate the LV.

Abstract 17706: Role of p62/SQSTM1 on Acute Ischemia-Induced Arrhythmias After Chronic Smoke in Mice

Introduction: Cigarette smoke presents a high-risk factor for coronary heart disease development. Cigarette smoke is often linked with increased susceptibility to cardiac ischemia and arrhythmias. p62/SQSTM1 is a stress-sensing protein responsible for proteasomal degradation that maintains redox homeostasis during cardiac tissue injury. However, its exact mechanism for arrhythmia development is not clear. Hypothesis: Here we tested the biological role of p62/SQSTM1 mice on cardiac ischemia injury-induced arrhythmogenesis after chronic cigarette smoke exposure. Methods: Mice (global p62/SQSTM1 deletion mice; p62/SQSTM1 -/- , 4 months old age) were exposed to cigarette smoke (9.4 mg tar/0.726 mg nicotine) for 16 weeks. Afterward, we utilized ischemia-reperfusion (IR) injury in intact hearts to test the risk of arrhythmias. Results: The smoke-exposed p62/SQSTM1 -/- isolated hearts (8 months old age) significantly reduced the incidence of ventricular arrhythmias compared to hearts obtained from p62/SQSTM1 +/+ mice post-IR during the first 15 minutes reperfusion (% of mice with ventricular arrhythmias, p62/SQSTM1 +/+ ; 7/10 hearts, p62/SQSTM1 -/- ; 2/8 hearts*, * <0.05). Abnormal Ca 2+ regulation in cardiomyocytes is triggering diastolic delayed afterdepolarizations and often leads to ventricular tachycardia. Therefore, we tested cellular Ca 2+ regulation with acute cigarette smoke extract (CSE) exposures in cardiomyocytes. p62/SQSTM1 -/- cardiomyocytes for 30 minutes exposed to CSE significantly alleviated CSE-induced spontaneous Ca 2+ waves (% of cardiomyocytes with spontaneous Ca 2+ waves, p62/SQSTM1 +/+ ; 6/13 cells, p62SQSTM1 -/- ; 1/15 cells*, * <0.05). Conclusions: Overall, our results suggest that the deletion of p62/SQSTM1 protein has a protective effect on acute cardiac ischemia-induced arrhythmogenesis after chronic cigarette smoke in mice.

A New Target of Electroacupuncture Pretreatment Mediated Sympathetic Nervous to Improve MIRI: Glutamatergic Neurons in Fastigial Nucleus of the Cerebellum

Ischemic heart disease is a fatal cardiovascular disease that irreversibly impairs the function of the heart, followed by reperfusion leading to a further increase in infarct size. Clinically, we call it myocardial ischemia–reperfusion injury (MIRI). A growing number of clinical observations and experimental studies have found electroacupuncture (EA) to be effective in alleviating MIRI. This study attempts to investigate whether glutamatergic neurons in fastigial nucleus (FN) of the cerebellum are involved in EA pretreatment to alleviate MIRI via sympathetic nerves, and the potential mechanisms of EA pretreatment process. A MIRI model was established by ligating the coronary artery of the left anterior descending branch of the heart for 30 minutes, followed by 2 hours of reperfusion. Multichannel physiological recordings, electrocardiogram, cardiac ultrasound, chemical genetics, enzyme-linked immunosorbent assay and immunofluorescence staining methods were combined to demonstrate that EA pretreatment inhibited neuronal firing and c-Fos expression in FN of the cerebellum and reduced cardiac sympathetic firing. Meanwhile, EA pretreatment significantly reduced cardiac ejection fraction (EF), shortening fraction (SF), percentage infarct area, decreased myocardial norepinephrine (NE), creatine kinase isoenzyme MB (CK-MB) concentrations, and improved MIRI-induced myocardial tissue morphology. The results were similar to the inhibition of glutamatergic neurons in FN. However, the activation of glutamatergic neurons in FN diminished the aforementioned effects of EA pretreatment. This study revealed that glutamatergic neurons in FN of the cerebellum is involved in EA pretreatment mediated sympathetic nervous and may be a potential mediator for improving MIRI.

Pre-treatment with pregabalin reduces liver ischemia-reperfusion injury in rats: tissue protection with an analgesic.

Ischemia-reperfusion injury is thought to be the most important factor affecting the success of liver surgery. Pregabalin has been studied to prevent ischemic reperfusion injury in many organs. The aim of this study was to investigate the role of pregabalin in preventing liver ischemic injury. 40 male Wistar-Albino rats, 6-8 weeks old, were divided into 5 groups. Four groups other than the sham group were subjected to hepatic ischemia for 1 hour, followed by 2 hours of reperfusion. Effects of 30 mg/and 60 mg/kg pregabalin were evaluated by aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor α (TNF-α), nuclear factor-kappa B (NF-кB), interleukin (IL)-6 levels, measured in blood samples collected before and after ischemia. Apoptosis was measured by caspase-3, and tissue samples were evaluated for ischemia by histopathologic examination. The 60 mg pregabalin group was significantly superior (p=0.024) to the N-acetylcysteine group and the 30 mg pregabalin group for AST levels (p=0.612 and p=0.807, respectively). The difference between before and after ischemia-reperfusion blood TNF-α levels was higher in the 60 mg pregabalin group, but not significantly different from the 30 mg pregabalin and N-acetylcysteine groups (p>0.05). Tissue TNF-α levels showed that 60 mg and 30 mg pregabalin treatment was more effective than no-treatment (p=0.011, p=0.033, respectively), but not superior to N-acetylcysteine (p>0.05). It has been found that ischemia-reperfusion causes damage to the liver, and this damage may be irreversible if no treatment is given. Our study group, pregabalin molecule was found to be significantly effective in preventing ischemia-reperfusion injury and may have a therapeutic advantage over N-acetylcysteine.

Osmundacetone alleviates cerebral ischemia-reperfusion injury in rats.

Osmundacetone (DHBAc) is an antioxidant compound that has been shown to have neuroprotective and immunomodulatory activities. However, few studies have estimated its effect on cerebral ischemia-reperfusion (I/R) injury. In this study, we investigated the protective effect of DHBAc on the brain tissue of rats with cerebral I/R injury. Rats were respectively given nimodipine (NI), low dose (L-DHBAc) and high dose (H-DHBAc) Osmundacetone, and they were killed under anesthesia after 24 hours of reperfusion. And neurological impairment scores, cerebral infarct size and cerebral pathological changes were respectively detected, and mRNA expression of recombinant kelch like ECH associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2), protein expression levels of caspase3, cleaved caspase3, heme oxygenase-1 (HO-1) and quinone oxidoreductase1 (NQO1) in ischemic brain tissue were measured. Compared with the I/R group, neurological impairment scores of the DHBAc groups were significantly decreased, and their infarct sizes were significantly smaller. DHBAc could improve the pathological status of brain tissue with cerebral I/R injury, including reducing number of inflammatory cells and area of vacuoles and restoring number of normal neurons. Expression levels of Keap1 mRNA and proteins of cleaved caspase3 were significantly decreased in the DHBAc groups than those of the I/R group, while expression levels of Nrf2 mRNA, HO-1 and NQO1 proteins were remarkably increased. The effect of H-DHBAc was similar to those of NI. These results suggest that DHBAc could mitigate damage to brain tissue in rats with cerebral I/R injury.

Cardioprotective Action of a Novel Synthetic 19,20-EDP Analog Is Sirt Dependent.

Mounting evidence suggests that cytochrome P450 epoxygenase-derived metabolites of docosahexaenoic acid, called epoxydocosapentaenoic acids (EDPs), limit mitochondrial damage after cardiac injury. In particular, the 19,20-EDP regioisomer has demonstrated potent cardioprotective action. Thus, we investigated our novel synthetic 19,20-EDP analog SA-22 for protection against cardiac ischemia-reperfusion (IR) injury. Isolated C57BL/6J mouse hearts were perfused through Langendorff apparatus for 20 minutes to obtain baseline function, followed by 30 minutes of global ischemia. Hearts were then treated with vehicle, 19,20-EDP, SA-22, or SA-22 with the pan-sirtuin inhibitor nicotinamide or the SIRT3-selective inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) at the start of 40 minutes reperfusion (N = 5-8). We assessed IR injury-induced changes in recovery of myocardial function, using left ventricular developed pressure and systolic and diastolic pressure change. Tissues were assessed for electron transport chain function, SIRT1 and SIRT3, optic atrophy type 1, and caspase-1. We also used H9c2 cells in an in vitro model of hypoxia/reoxygenation injury (N = 3-6). Hearts perfused with SA-22 had significantly improved postischemic left ventricular developed pressure, systolic and diastolic recovery (64% of baseline), compared with vehicle control (15% of baseline). In addition, treatment with SA-22 led to better catalytic function observed in electron transport chain and SIRT enzymes. The protective action of SA-22 resulted in reduced activation of pyroptosis in both hearts and cells after injury. Interestingly, although nicotinamide cotreatment worsened functional outcomes, cell survival, and attenuated sirtuin activity, it failed to completely attenuate SA-22-induced protection against pyroptosis, possibly indicating EDPs exert cytoprotection through pleiotropic mechanisms. In short, these data demonstrate the potential of our novel synthetic 19,20-EDP analog, SA-22, against IR/hypoxia-reoxygenation injury and justify further development of therapeutic agents based on 19,20-EDP.

Hepatoprotective effects of linalool against liver ischemia-reperfusion: the role of Nrf2/HO-1/NQO1 and TLR4/RAGE/NFκB pathways.

Hepatic ischemia-reperfusion (H I/R) injury arises due to a temporary obstruction followed by the re-establishment of blood supply to the liver. Linalool (LIN), a main volatile constituent of essential oils in numerous aromatic plant species, exhibited various medicinal and pharmacological actions. This study investigated the protective effect of LIN on the status of H I/R, with the study of the possible mechanisms. In addition, linalool's antagonistic effects were tested against several metabolic targets using in silico molecular docking technique. Wistar rats were allocated into five groups. Sham and LIN + Sham groups in which animals were administered either vehicle (1% CMC) or LIN (200 mg/kg/day) orally for two weeks. H I/R group in which rats were administered 1% CMC for two weeks and then experienced hepatic ischemia for 60 min followed by 6 hours of reperfusion. LIN 100 + H I/R and LIN 200 + H I/R groups in which rats were pretreated with LIN (100, 200 mg/kg/day) respectively for two weeks, then subjected to H I/R. H I/R-induced injury resulted in impaired liver function and activated Keap1/Nrf2/HO-1/NQO1 and HMGB1/TLR4/RAGE/NFкB pathways with subsequent oxidative stress, inflammation, and apoptosis. LIN pretreatment alleviated I/R-induced impairment in liver function, promoted Keap1/Nrf2/HO-1/NQO1, and mitigated the HMGB1/TLR4/RAGE/NFкB pathway. LIN pre-administration deterred adhesion molecule, neutrophils infiltration, RAGE, IL-1β, IL-6, TNF-α, and apoptosis. LIN demonstrated hepato-protective effects against H I/R via instigation Keap1/Nrf2/HO-1/NQO1 and mitigating the HMGB1/TLR4/RAGE/NFкB pathways with subsequent deterring oxidative stress, inflammation, and apoptosis.

Neuroprotective effects of Coenzyme Q10 in ischemia-reperfusion injury via inflammation and oxidative stress reduction in adult male rats.

This study aimed to investigate the potential neuroprotective effects of coenzyme Q10 in cerebral ischemia-reperfusion injury-induced neuronal damage and explore the underlying mechanisms. Twenty-eight adult male rats, weighing approximately 200-300 grams, were randomly divided into four groups: the sham group (neck dissection without ischemia), the control group (30 minutes of bilateral common carotid artery ligation followed by one hour of reperfusion), the vehicle group (oral carboxymethylcellulose solution for seven days prior to bilateral common carotid artery ligation and reperfusion), and the treatment group (seven days of coenzyme Q10 pretreatment followed by bilateral common carotid artery occlusion and reperfusion). Histopathological analysis and measurement of brain infarct size were performed, and cerebral levels of IL-6, IL-10, TNF-α, ICAM-1, NF-κB p65, and total antioxidant capacity were assessed. These cerebral tissue levels and cerebral infarct size were significantly elevated in the control and vehicle groups compared to the sham group. Conversely, the total antioxidant capacity was significantly reduced in these groups. Coenzyme Q10 treatment resulted in a significant increase in IL-10 and total antioxidant capacity levels, along with a significant decrease in IL-6, ICAM-1, TNF-α, and NF-κB p65 levels. Histopathological analysis revealed a significant reduction in ischemic damage in the coenzyme Q10-treated group. Coenzyme Q10 has neuroprotective properties in rats subjected to cerebral ischemia/reperfusion injury, possibly through its anti-inflammatory and anti-oxidative effects.

Leucine Reduced Blood-Brain Barrier Disruption and Infarct Size in Early Cerebral Ischemia-Reperfusion.

A disruption of the blood-brain barrier (BBB) is a crucial pathophysiological change that can impact the outcome of a stroke. Ribosomal protein S6 (S6) and protein kinase B (Akt) play significant roles in early cerebral ischemia-reperfusion injury. Studies have suggested that branched-chain amino acids (BCAAs) may have neuroprotective properties for spinal cord or brain injuries. Therefore, we conducted research to investigate if leucine, one of the BCAAs, could offer neuroprotection and alter BBB disruption, along with its effects on the phosphorylation of S6 and Akt during the early phase of cerebral ischemia-reperfusion, specifically within the thrombolytic therapy time window. In rats, ten min after left middle cerebral artery occlusion (MCAO), 5 µL of 20 mM L-leucine or normal saline was injected into the left lateral ventricle. After two hours of reperfusion following one hour of MCAO, we determined the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid to assess the BBB disruption, infarct size, and phosphorylation of S6 and Akt. Ischemia-reperfusion increased the Ki (+143%, p < 0.001) and the intra-cerebroventricular injection of leucine lowered the Ki in the ischemic-reperfused cortex (-34%, p < 0.001). Leucine reduced the percentage of cortical infarct (-42%, p < 0.0001) out of the total cortical area. Ischemia-reperfusion alone significantly increased the phosphorylation of both S6 and Akt (p < 0.05). However, the administration of leucine had no further effect on the phosphorylation of S6 or Akt in the ischemic-reperfused cortex. This study suggests that an acute increase in leucine levels in the brain during early ischemia-reperfusion within a few hours of stroke may offer neuroprotection, possibly due to reduced BBB disruption being one of the major contributing factors. Leucine did not further increase the already elevated phosphorylation of S6 or Akt by ischemia-reperfusion under the current experimental conditions. Our data warrant further studies on the effects of leucine on neuronal survival and its mechanisms in the later stages of cerebral ischemia-reperfusion.

Neuroprotective effects of a hemoglobin-based oxygen carrier (stroma-free hemoglobin nanoparticle) on ischemia reperfusion injury

The application of hemoglobin (Hb)-based oxygen carriers (HBOCs) to the treatment of cerebral ischemia has been investigated. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA3) was found to exert neuroprotective effects on ischemia/reperfusion injury. Stroma-free hemoglobin nanoparticles (SFHbNP), a subsequently developed HBOC consisting of a spherical polymerized stroma-free Hb core with a HSA shell, contains the natural antioxidant enzyme catalase and, thus, is expected to exert additive effects. We herein investigated whether SFHbNP exerted enhanced neuroprotective effects in a rat transient middle cerebral artery occlusion (tMCAO) model. Rats were subjected to 2-hour tMCAO and divided into the following 3 groups with the intravenous administration of the respective reagents: (1) phosphate-buffered saline (PBS), as a vehicle (2) Hb-HSA3, and (3) SFHbNP. After 24-hour reperfusion, infarct and edema volumes decreased in the order of the PBS, Hb-HSA3, and SFHbNP groups, with a significant difference (p < 0.05) between the PBS and SFHbNP groups. Similar reductions were observed in oxidative stress, leukocyte recruitment, and blood–brain barrier disruption in the order of the PBS, Hb-HSA3, and SFHbNP groups. In the early phase of reperfusion within 6 h, microvascular HBOC perfusion and cerebral blood flow were maintained at high levels during the reperfusion period in the Hb-HSA3 and SFHbNP groups. However, a difference was observed in tissue oxygen partial pressure levels, which significantly decreased after 6-hour reperfusion in the Hb-HSA3 group, but remained high in the SFHbNP group. A superior oxygen transport ability appears to be related to the enhanced neuroprotective effects of SFHbNP.

Metabolic Tissue Swelling and Local Microcirculation in Splanchnic Artery Occlusion Shock: Implications for Critical Illness.

Trauma is a leading cause of death in the United States. Advancements in shock resuscitation have been disappointing because the correct upstream mechanisms of injury are not being targeted. Recently, significant advancements have been shown using new cell-impermeant molecules that work by transferring metabolic water from swollen ischemic cells to the capillary, which restores tissue perfusion by microcirculatory decompression. The rapid normalization of oxygen transfer improves resuscitation outcomes. Since poor resuscitation and perfusion of trauma patients also causes critical illness and sepsis and can be mimicked by ischemia-reperfusion of splanchnic tissues, we hypothesized that inadequate oxygenation of the gut during trauma drives development of later shock and critical illness. We further hypothesized that this is caused by ischemia-induced water shifts causing compression no-reflow. To test this, the superior mesenteric artery of juvenile anesthetized swine was occluded for 30 minutes followed by 8 hours of reperfusion to induce mild splanchnic artery occlusion (SAO) shock. One group received the impermeant polyethylene glycol 20,000 Da (PEG-20k) that prevents metabolic cell swelling, and the other received a lactated Ringer's vehicle. Survival doubled in PEG-20k-treated swine along with improved macrohemodynamics and intestinal mucosal perfusion. Villus morphometry and plasma inflammatory cytokines normalized with impermeants. Plasma endotoxin rose over time after reperfusion, and impermeants abolished the rise. Inert osmotically active cell impermeants like PEG-20k improve intestinal reperfusion injury, SAO shock, and early signs of sepsis, which may be due to early restoration of mucosal perfusion and preservation of the septic barrier by reversal of ischemic compression no-reflow. SIGNIFICANCE STATEMENT: Significant advancements in treating shock and ischemia have been disappointing because the correct upstream causes have not been targeted. This study supports that poor tissue perfusion after intestinal ischemia from shock is caused by capillary compression no-reflow secondary to metabolic cell and tissue swelling since selectively targeting this issue with novel polyethylene glycol 20,000 Da-based cell-impermeant intravenous solutions reduces splanchnic artery occlusion shock, doubles survival time, restores tissue microperfusion, and preserves gut barrier function.

Destabilization of the Organized Structure of Ventricular Fibrillation During Reperfusion

Aim: to study the effect of reperfusion on the organized frequency-amplitude structure of ventricular fibrillation (VF) in the dog heart.Materials and methods. We conducted 4 experiments on 8 dogs. In each experiment, the isolated heart of one dog was perfused with the blood of the second (supporting) dog. In 4 experiments on an isolated artificially perfused heart, 6 episodes of 3 min ischemia and 10 min reperfusion of the heart were performed in VF (1–2 episodes of ischemia-reperfusion in one experiment). Each episode of 3 min ischemia in VF was preceded by a 10 min perfusion of the heart in VF. Ventricular electrogram was recorded during VF episodes. A frequencyamplitude (spectral) analysis of 1 sec segments of the electrogram was performed, and the proportion (in %) of 0.5–15 Hz frequency oscillations in 10 sec segments of the electrogram was determined in 6 episodes of perfusion, ischemia and reperfusion in VF (M±m, N=60). The VF frequency-amplitude structures during ischemia and reperfusion were compared with the stable VF frequency-amplitude structure during perfusion taken as the control. The nonparametric Welch criterion in the «The R Project for Statistical Computing» software environment was used to compare the VF parameters during perfusion, ischemia and reperfusion. Results. 9–10 Hz frequency oscillations dominated in the VF frequency-amplitude structure during heart perfusion, taken as the control. In the first 30 sec of ischemia, the frequency and amplitude of the dominant oscillations did not significantly change vs VF control obtained during cardiac perfusion. A decrease of dominant oscillations frequency up to 6.5–7.5 Hz, and of the proportion of oscillations — up to 26% was documented at the 3rd min of ischemia. At the 1st min of reperfusion, the frequency of dominant oscillations increased to 13.5–14.5 Hz, but the proportion of oscillations remained reduced to 26%, as at the 3rd min of ischemia. At the 2nd min of reperfusion, the frequency of dominant oscillations decreased to 9.5–10.5 Hz, and the proportion of dominant oscillations increased to 33%. The frequency and amplitude of the dominant oscillations stabilized at 3–10 min of reperfusion: oscillations at 9–10 Hz frequency accounted for 32–33% of the spectral power.Conclusion. Reperfusion in VF is characterized by transient destabilization of VF organized structure at the 1st min of the procedure. VF organized structure regains stabilization within 2–10 min of reperfusion. Cardiac perfusion in intentionally induced VF can be used instead of cardioplegia during major cardiac surgery to boost cardiac resistance to ischemia and prevent or reduce reperfusion complications.

Prophylactic Treatment of Intestinal Ischemia-Reperfusion Injury Reduces Mucosal Damage and Improves Intestinal Absorption.

Intestinal ischemia-reperfusion injury (i-IRI) involves a blood flow interruption in an intestinal segment followed by blood flow restoration. When blood flow is restored, oxidative and inflammatory molecules are distributed throughout the bloodstream, triggering both local and systemic damage. Our goal was to evaluate the potential of three antioxidant and/or anti-inflammatory compounds (curcumin, dexmedetomidine and α-tocopherol) to prevent or reverse local and systemic damage induced by i-IRI. i-IRI was induced by placing a microvascular clip in the superior mesenteric artery of female WAG/RijHsd rats; the clip was removed after 1h and reperfusion was allowed for 4h. Curcumin (200 mg/kg, orally), α-tocopherol (20 mg/kg, i.p.), and dexmedetomidine (5 or 20 µg/kg, s.c.; DEX5 and DEX20, respectively) were administered. Blood and terminal ileum specimens were collected for biochemical and histological determination. Furthermore, D-xylose absorption test was performed to evaluate intestinal absorption; after completing the 1-hour ischemia and 4-hour reperfusion period, 1 mL of aqueous D-xylose solution (0.615 mg/mL) was administered orally, and one hour later, plasma D-xylose levels were quantified. The histological injury degree (HID) measured by the Chiu scale was significantly reduced when the treatments were applied (non-treated rats, 2.6 ± 0.75; curcumin, 1.54 ± 0.8; DEX5, 1.47 ± 0.7; DEX20 1.14 ± 0.5; and α-tocopherol, 1.01 ± 0.6); intestinal absorptive capacity also improved in all cases healthy rats (2.06 ± 0.07 µg/mL; non-treated, 1.18 ± 0.07 µg/mL; curcumin 1.76 ± 0.3 µg/mL; DEX5, 2.29 ± 0.2 µg/mL; DEX20, 2.25 ± 0.26 µg/mL; and α-tocopherol 1.66 ± 0.21 µg/mL). However, it failed to reduce liver enzyme levels. Finally, only dexmedetomidine significantly reduced urea and creatinine levels compared to non-treated animals. All drugs were effective in reducing HID, although α-tocopherol was effective to a greater extent. Only dexmedetomidine reverted intestinal absorption to normal values of healthy animals.

Erratum: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation.

Erratum: Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation.

Properdin inhibition ameliorates hepatic ischemia/reperfusion injury without interfering with liver regeneration in mice.

Hepatic ischemia/reperfusion injury (IRI) often causes serious complications in liver surgeries, including transplantation. Complement activation seems to be involved in hepatic IRI; however, no complement-targeted intervention has been clinically applied. We investigated the therapeutic potential of Properdin-targeted complement regulation in hepatic IRI. Male wild-type mice (B10D2/nSn) were exposed to 90-minute partial hepatic IRI to the left and median lobes with either monoclonal anti-Properdin-antibody (Ab) or control-immunoglobulin (IgG) administration. Since the complement system is closely involved in liver regeneration, the influence of anti-Properdin-Ab on liver regeneration was also evaluated in a mouse model of 70% partial hepatectomy. Anti-Properdin-Ab significantly reduced serum transaminases and histopathological damages at 2 and 6 hours after reperfusion (P <0.001, respectively). These improvements at 2 hours was accompanied by significant reductions in CD41+ platelet aggregation (P =0.010) and ssDNA+ cells (P <0.001), indicating significant amelioration in hepatic microcirculation and apoptosis, respectively. Characteristically, F4/80+ cells representing macrophages, mainly Kupffer cells, were maintained by anti-Properdin-Ab (P <0.001). Western blot showed decreased phosphorylation of only Erk1/2 among MAPKs (P =0.004). After 6 hours of reperfusion, anti-Properdin-Ab significantly attenuated the release of HMGB-1, which provokes the release of proinflammatory cytokines/chemokines (P =0.002). Infiltration of CD11b+ and Ly6-G+ cells, representing infiltrating macrophages and neutrophils, respectively, were significantly alleviated by anti-Properdin-Ab (both P <0.001). Notably, anti-Properdin-Ab did not affect remnant liver weight and BrdU+ cells at 48 hours after 70% partial hepatectomy (P =0.13 and 0.31, respectively). In conclusion, Properdin inhibition significantly ameliorates hepatic IRI without interfering with liver regeneration.

Use of peroxiredoxin 6 to prevent liver dysfunction in acute kidney injury

Acute kidney injury causes deterioration of liver function, that is a confounding factor affecting treatment outcomes. In this work, renal ischemia reperfusion injury was used as a model. Taking into account that hyperproduction of reactive oxygen species is the major risk factor for kidney damage, the exogenous antioxidant enzyme peroxiredoxin 6, able to neutralize reactive oxygen species, has been used to prevent liver damage when kidneys are damaged. Kidney injury was initiated by a 45-minute ischemia simultaneously with a left-sided donor nephrectomy without manipulations of the liver. Peroxiredoxin 6 was administered intravenously 15 minutes before ischemia. The functional state of the liver was assessed after 2, 5 and 24 hours of reperfusion using histological and biochemical analysis. The signs of liver damage were detected in the best possible way after 5 hours of kidney reperfusion. It was found that peroxiredoxin 6 helps reduce the severity of the vascular reaction and leukocyte infiltration in the liver; lower the level of dystrophy and apoptosis of hepatocytes; keep the concentration of TBA-reactive products even and stabilize the level of cytokines, IL-6 and IL-10, in the liver tissue, as well as normalize the activity of intracellular transferases in the blood at the onset of reperfusion. The protective effect of peroxiredoxin 6 is associated primarily with its antioxidant properties, due to which hyperproduction of reactive oxygen species can be neutralized in the early phase of kidney reperfusion, but the signal-regulatory function of the protein can also contribute to a protective role peroxiredoxin 6.

Mitsugumin 53 mitigation of ischemia–reperfusion injury in a mouse model

ObjectivePrimary graft dysfunction is often attributed to ischemia–reperfusion injury, and prevention would be a therapeutic approach to mitigate injury. Mitsugumin 53, a myokine, is a component of the endogenous cell membrane repair machinery. Previously, exogenous administration of recombinant human (recombinant human mitsugumin 53) protein has been shown to mitigate acute lung injury. In this study, we aimed to quantify a therapeutic benefit of recombinant human mitsugumin 53 to mitigate a transplant-relevant model of ischemia–reperfusion injury. MethodsC57BL/6J mice were subjected to 1 hour of ischemia (via left lung hilar clamp), followed by 24 hours of reperfusion. mg53−/− mice were administered exogenous recombinant human mitsugumin 53 or saline before reperfusion. Tissue, bronchoalveolar lavage, and blood samples were collected at death and used to quantify the extent of lung injury via histology and biochemical assays. ResultsAdministration of recombinant human mitsugumin 53 showed a significant decrease in an established biometric profile of lung injury as measured by lactate dehydrogenase and endothelin-1 in the bronchoalveolar lavage and plasma. Biochemical markers of apoptosis and pyroptosis (interleukin-1β and tumor necrosis factor-α) were also significantly mitigated, overall demonstrating recombinant human mitsugumin 53's ability to decrease the inflammatory response of ischemia–reperfusion injury. Exogenous recombinant human mitsugumin 53 administration showed a trend toward decreasing overall cellular infiltrate and neutrophil response. Fluorescent colocalization imaging revealed recombinant human mitsugumin 53 was effectively delivered to the endothelium. ConclusionsThese data demonstrate that recombinant human mitsugumin 53 has the potential to prevent or reverse ischemia–reperfusion injury–mediated lung damage. Although additional studies are needed in wild-type mice to demonstrate efficacy, this work serves as proof-of-concept to indicate the potential therapeutic benefit of mitsugumin 53 administration to mitigate ischemia–reperfusion injury.

Abstract P2153: A Long Noncoding RNA Plentiful In Cardiosphere-derived Cell Extracellular Vesicles (CDC-EVs), BCYRN1, And Its Short Synthetic Derivatives Enhance Regulatory T Cell (Treg) Proliferation, Migration, And Activation

Background: By modulating inflammation, Treg cells influence outcomes in myocardial infarction (MI). Here, we test the hypothesis that BCYRN1 (Brain Cytoplasmic RNA 1) and BCYRN1-derived binding small sequences (BDSSs) are cardioprotective by activating Tregs. Methods: RNA sequencing identified BCYRN1 as plentiful in cardiosphere-derived cell extracellular vesicles (CDC-EVs), validated by qPCR. RNA pulldown and luciferase assays probed BCYRN1's potential function as a miRNA sponge. We assessed BCYRN1 and BDSS effects on Treg proliferation (Cell Counting Kit-8), migration (trans-well migration), and IL-10 (qPCR and ELISA). In vivo , MI was induced in C57BL/6 mouse by left coronary artery ligation for 45 minutes followed by 15 minutes reperfusion. Afterwards, mice received (via tail vein) CDC-EV-BCYRN1 (CDC-EVs overexpressing BCYRN1), BDSSs, mut-BDSSs (scrambled inactive versions of BDSS) or vehicle (PBS). MI size (by TTC staining) and infiltrating Treg (by flow cytometry) were assessed 72 hrs post-MI. Findings: BCYRN1 was found to contain putative binding sites for miR-138, miR-150 and miR-98; these miRs target ATG7, CCR6 and IL-10 mRNAs, respectively. BCYRN1 serves as a miRNA sponge, leading to Treg cell ATG7/autophagy dependent proliferation (3.0± 0.1 fold increase, N=5), CCR-6 dependent migration (1.8 ± 0.1 fold increase, N=5) and increased IL-10 production (fold increase, qPCR: 2.6 ± 0.4, N=6 and ELISA: 6.1 ± 0.8, N=5). Lipid nanoparticles containing a cocktail of 3BDSSs likewise induce Treg cell proliferation (1.9± 0.6 fold increase, N=6), migration (1.3± 0.2 fold increase, N=6), and IL-10 induction (6.8± 0.5 fold increase, N=6). In vivo (N=5/group), CDC-EV-BCYRN1 (but not vehicle) and BDSS cocktail (but not mut-BDSS) induced Treg infiltration, proliferation and IL10 production in the heart with a concomitant decrease of infarct size. Conclusions: A single LncRNA (BCYRN1) and shorter synthetic derivatives mimic the effects of CDC-EV on Treg in vitro and in vivo . Our study provides a novel approach, targeting Treg, to treat MI. This approach could be useful for other diseases where increased Treg activity is a therapeutic goal (e.g. autoimmune disorders).