Minutes Of Reperfusion Research Articles

Comparison of hemodynamic and infarct-limiting effects of preservation solution based on Krebs–Henseleit buffer and HTK solution in the rat model of heterotopic heart transplantation

Objective. To compare the cardioprotective effcacy of a preservation solution based on Krebs–Henseleit buffer and HTK solution in the model of heterotopic heart transplantation in rat.Design and methods. A study was conducted on 12 Wistar rats. The animals were divided into groups depending on the preservation solution used: 1) Krebs–Henseleit buffer-based solution (n = 7), 2) HTK (n = 5). Each experiment consisted of collecting donor heart, preserving it with an appropriate cardioplegic solution, heterotopic transplantation into a recipient rat followed by explantation and evaluation of left ventricular contractility using the Langendorff model and histochemical assessment of the irreversible myocardial damage. Coronary blood flow in the donor heart was assessed in vivo using ultrasound doppler flowmetry. After 3 hours, the donor heart was explanted and connected to the Langendorff apparatus to assess left ventricular contractility, and the myocardium was subjected to histochemical staining with 1 % triphenyltetrazolium chloride for the assessment of the irreversible myocardial damage.Results. In the group of Krebs–Henseleit buffer-based cardioplegic solution, 7 experiments were performed. Myocardial infarct size was 3,5 ± 1,2%, the coronary flow rate was 4,5 ± 1,3 ml/min, and the developed left ventricular pressure of the donor heart was 70 ± 6,3 mmHg at diastolic left ventricular pressure of 10 mmHg. In the HTK solution group (n = 5), in all of the experiments after the start of blood flow, the transplanted heart did not begin to contract, and all 5 hearts remained in an asystole state. Therefore, after the end of the 3-hour reperfusion period, the assessment of the contractility of the left ventricle using the Langendorff apparatus was not carried out. Coronary flow rate was only 0,4 ± 0,1 ml/min, probably resulting from inadequate cardioprotection with HTK solution. Due to the lack of adequate reperfusion, the accurate assessment of the irreversible myocardial damage was impossible in the HTK solution group.Conclusions. The technique of heterotopic heart transplantation in rats is adequate and informative for the study of ischemia-reperfusion myocardial damage as well as for the study of the effectiveness of cardioplegic and cardiac preservation solutions. Cardioplegic solution based on Krebs– Henseleit buffer demonstrated greater cardioprotective effectiveness in our model compared to HTK solution.

Cardioprotective effects of a synthetic peptide targeting the extrinsic apoptotic pathway in a mouse model of ischemia-reperfusion

Apoptosis is a main contributor of myocardial reperfusion injury during acute infarction. In a previous study, we showed that reperfusion injury was mediated by the FAS-dependent apoptotic signal mobilizing the DAXX (death-associated protein) adaptor protein. To evaluate the cardioprotective effects of a synthetic peptide that uncouples FAS to the DAXX downstream pathway. The SPOT technology was used to design a synthetic peptide interfering with FAS:DAXX interaction. This peptide was coupled to the Tat cell penetrating peptide (Tat-DAXXp). Infarct size (TTC staining) and apoptosis (DNA fragmentation) were evaluated in the left ventricle of mice subjected to a surgical protocol of reversible coronary artery ligation and treated by the peptide at the onset of reperfusion. Cellular internalization of the peptide was measured using a fluorescent peptide both in primary cultures of cardiomyocytes or in left ventricles after surgery. Anti-apoptotic properties of the synthetic peptide were demonstrated in primary cardiomyocytes. In vivo, one bolus of Tat-DAXXp (1 mg/kg), injected intravenously 5 min before reperfusion in a murine myocardial ischemia-reperfusion model decreased infarct size by 48% after 24 hours of reperfusion. After a 30-min delayed administration, Tat-DAXXp was still able to protect against reperfusion-induced apoptosis and was completely degraded and eliminated within 24 hours thereby reducing risks of potential side effects. Importantly, post-infarction mortality was reduced by 67% by Tat-DAXXp treatment. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting ischemia-reperfusion injury.

Effect of hyperoxic reperfusion on final infarct size after ST elevation myocardial infarction (STEMI) in old Wistar rats

Elderly people above 65 years old account for more than 80% of patients with ischemic heart disease [1] . Most of animal models of myocardial ischemia-reperfusion injury (IRI) involve young animals, with the assumption that findings are applicable to older animals and thus applicable to humans at highest risk, the elderly. Current studies failed to analyse the effect of hyperoxia during myocardial infarction (MI) reperfusion in old rodent model. Assess the final myocardial infarct size after normoxic and hyperoxic MI reperfusion in old Wistar rats. STEMI was induced in old male Wistar rats (35–40 weeks old) by ligation of the left anterior descending (LAD) artery and maintained for 60 minutes. Animals were randomised either in the hyperoxic reperfusion (HR) group (receiving 100% oxygen) or the normoxic reperfusion (NR) group (21% of oxygen) for a 3-hour reperfusion. At the end of reperfusion, Evans Blue dye solution was injected to delineate the ischemic area from the non-ischemic area. The heart was cut into transverse slices and immerged in TTC solution. Myocardial infarct size was assessed in two ways; first troponin T concentration was measured after reperfusion, secondly ischemic zone and area at risk of the myocardium slices were measured by planimetry. A total of 8 rats underwent HR vs 11 NR. No difference was observed in the final myocardial infarct size between the two groups in both methods of myocardial infarct size assessment. These very preliminary results corroborate the observations made in young animals’ studies [2] , [3] , however they should be taken cautiously since they are based on a very small cohort. There is a need to understand the effect of hyperoxic MI reperfusion as animal studies contradict the findings in human studies [4] .

The Effect of Hepcidin on Cardiac Ischemia-Reperfusion Injury

Background/aim: Hepcidin is the main hormone in the regulation of iron metabolism which is also released from the heart. The aim of our study was to investigate the effects of hepcidin on the cardiac ischemia-reperfusion injury.Materials and methods: In this study, 12 Wistar albino rats were divided into two groups (n = 6 each): 1) The ischemia-reperfusion group (Group 1); 2) Hepcidin-treated group (Group 2). Rat hearts were perfused on Langendorff system with KH (Krebs-Henseleit) and subjected to 30 min stabilization, 30 min global ischemia, and 30 min reperfusion. Hepcidin (- M) was applied to group 2 at the onset of ischemia. Malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NOx) levels were measured in heart tissue for NOx levels, viscosity, and ion content of perfusate were collected before ischemia and the 1st, 5th, 10th, 20th, and 30th minutes of reperfusion were determined. Apoptosis in heart was evaluated.Results: NOx and MDA levels significantly decreased in heart tissue in Hepcidin-treated group. NOx and viscosity of perfusate were not significantly different between the groups. Perfusate iron, calcium, magnesium, potassium, and sodium levels in group 2 were more homogeneous. Histologic structures of heart tissue were regularly in group 2. Apoptosis were increased in control group compared to hepcidin treated group.Conclusion: These results suggest that hepcidin may have a protective effect on the heart for the ischemia-reperfusion injury.

Effects of Conivaptan versus Mannitol on Post-Ischemic Brain Injury and Edema.

The aim of this study was to compare the effects of conivaptan, an arginine vasopressin antagonist, and mannitol, a sugar alcohol, on cerebral ischemia-induced brain injury and edema in rats. Fifty-eight 8-week-old male Sprague Dawley rats were randomly divided into five groups: control, ischemia-reperfusion (I/R)+saline, I/R+mannitol, I/R+10 mg/ml conivaptan, and I/R+20 mg/ml conivaptan. Cerebral ischemia was induced by common carotid artery occlusion for 30 minutes. Saline, mannitol, or conivaptan were administered intravenously at the onset of reperfusion. Blood and brain tissue samples were taken at the 6th hour of reperfusion. The electrolytes (Na+-K+-Cl-), osmolality, arginine vasopressin, albumin, progranulin (PGRN), neuron-specific enolase (NSE), and myeloperoxidase activity were measured in rat serum samples. Brain frontal/hippocampal sections were stained with hematoxylin-eosin and TUNEL techniques to evaluate histopathological changes. Statistical analyses revealed that conivaptan caused significant changes in the electrolyte, NSE, and PGRN levels and osmolality when compared with mannitol. Conivaptan treatment showed positive effects on serum biochemistry and tissue histology. Our findings revealed that conivaptan shows more diuretic activity than mannitol and triggers neither any damages nor edema in the brain tissue. This study may provide beneficial information for the development of treatment strategies for ischemia-related cerebrovascular diseases.

The Effect of 30-minute Ischemia on the Levels of IL6, TNFα, NO, Glutathione and Lactic Acid in the Hepatic Tissue of Rats with Hepatic Steatosis

Fatty liver disease (FLD) is becoming increasingly common. Ischemia-reperfusion injury (IRI) is the main pathogenic factor determining morbidity and mortality after major hepatic surgery. This experimental study investigated changes in the levels of interleukin 6 (IL6), tumor necrosis factor α (TNFα), nitric oxide (NO), glutathione (GSH) and lactic acid (LA) in hepatic tissue, in normal rats and rats with severe hepatic steatosis, after 30-minute hepatic ischemia and reperfusion. The aim was to evaluate the ability of the severely steatotic liver to sustain ischemia. The study was conducted on 96 male Wistar rats aged 12–14 weeks, in 48 of which severe hepatic steatosis was induced by administration of a choline-free diet for 12–14 weeks. The other 48 rats received standard laboratory diet for the same period. The effect of 30-minute hepatic ischemia and reperfusion was examined in 24 steatotic and 24 lean livers, which were subjected to hepatic ischemia-reperfusion injury. After 6 and 24 hours of reperfusion, the levels of IL6, TNFα, NO, LA and GSH in the liver tissue of all the rats were measured. The serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were measured to determine the severity of hepatic injury, and the survival of the rats after surgery was assessed. Rats with steatotic livers subjected to 30-minute hepatic ischemia and reperfusion showed significantly higher liver values of TNFα and LA and significantly lower liver values of IL6, GSH and NO than lean rats. The serum levels of AST and ALT were higher in rats with severely steatotic livers than in those with lean livers, after 30-minute hepatic ischemia and reperfusion, and no rat with a steatotic liver survived surgery. Rats with steatotic livers undergoing 30-minute ischemia and reperfusion show significant elevation of TNFα and LA and reduction in IL6, GSH and NO in liver tissue, with poor survival after surgery, compared to non-steatotic rats. These findings suggest that steatotic livers are more susceptible to ischemia-reperfusion injury than lean livers.

Cardioprotection by Humoral Factors Released After Remote Ischemic Preconditioning Depends on Anesthetic Regimen.

Remote ischemic preconditioning (RIPC) is a practicable and noninvasive method to protect the heart against ischemia reperfusion injury. Unfortunately results from clinical studies are not convincing. Propofol is suggested to be an inhibiting factor of cardioprotection by RIPC, but the underlying mechanism is still unknown. We investigated whether after RIPC the release of humoral factors and/or the direct cardioprotective effect at the myocardium is inhibited by propofol. Randomized, prospective, blinded laboratory investigation. Experimental laboratory. Male Wistar rats. Repetitive hind limb ischemia in rats-blood plasma transfers to isolated rat heart. In male Wistar rats (six groups, each n = 6/group), RIPC was induced by four cycles of 5 minutes bilateral hind limb ischemia alternately with 5 minutes of reperfusion. Blood samples were taken with (RIPC) and without RIPC (Con). Rats received continuous anesthesia with pentobarbital (Pento, 40 mg/kg body weight/hr) or propofol (Prop, 12 mg/kg body weight/hr), respectively. Cardioprotective properties of the blood plasma was investigated in the rat heart in vitro (six groups, each n = 6/group) perfused with Krebs-Henseleit buffer alone or with propofol (10 µM). Plasma was administered over 10 minutes before myocardial ischemia. All hearts underwent 33 minutes of global ischemia followed by 1 hour of reperfusion. At the end of the experiments, infarct size was determined by triphenyl-tetrazolium-chloride staining. RIPC plasma from pentobarbital anesthetized rats (Pento-RIPC) reduced infarct size from 64% (62-71%) (Pento-Con) to 34% (30-39%) (p < 0.0001). Infarct size with control plasma from propofol anesthetized rats was 59% (58-64%) (Prop-Con). RIPC plasma could not induce cardioprotection (Prop-RIPC: 63% [56-70%] ns vs Prop-Con). In contrast, RIPC plasma from pentobarbital anesthetized rats induced a significant infarct size reduction under propofol perfusion (Pento-RIPC: 34% [30-42%] vs Pento-Con: 54% [53-63%]; p < 0.0001). Loss of cardioprotection by RIPC during propofol anesthesia depends on inhibition of release of humoral factors.

Emodin alleviates myocardial ischemia/reperfusion injury by inhibiting gasdermin D-mediated pyroptosis in cardiomyocytes.

BackgroundEmodin has recently been reported to have a powerful antiinflammatory effect, protecting the myocardium against ischemia/reperfusion (I/R) injury. Pyroptosis is a proinflammatory programmed cell death that is related to many diseases. The present study investigated the effect of emodin on pyroptosis in cardiomyocytes.Materials and methodsSprague Dawley rats were randomly divided into sham, I/R, and I/R+Emodin groups. I/R model was subjected to 30 minutes’ ligation of left anterior descending coronary artery, followed by 2 hours of reperfusion. Cardiomyocytes were exposed to hypoxic conditions for 1 hour and normoxic conditions for 2 hours. The level of the pyroptosis was detected by Western blot, real-time PCR analysis, and ELISA.ResultsThe level of gasdermin D-N domains was upregulated in cardiomyocytes during I/R or hypoxia/reoxygenation (H/R) treatment. Moreover, emodin increased the rate of cell survival in vitro and decreased the myocardial infarct size in vivo via suppressing the levels of I/R-induced pyroptosis. Additionally, the expression of TLR4, MyD88, phospho-IκBα, phospho-NF-κB, and the NLRP3 inflammasome was significantly upregulated in cardiomyocytes subjected to H/R treatment, while emodin suppressed the expression of these proteins.ConclusionThis study confirms that emodin treatment was able to alleviate myocardial I/R injury and inhibit pyroptosis in vivo and in vitro. The inhibitory effect of emodin on pyroptosis was mediated by suppressing the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, emodin may provide an alternative treatment for myocardial I/R injury.

DNAX Activating Protein of 12 kDa/Triggering Receptor Expressed on Myeloid Cells 2 Expression by Mouse and Human Liver Dendritic Cells: Functional Implications and Regulation of Liver Ischemia-Reperfusion Injury.

Liver interstitial dendritic cells (DCs) have been implicated in the control of ischemia-reperfusion injury (IRI) and host immune responses following liver transplantation. Mechanisms underlying these regulatory functions of hepatic DCs remain unclear. We have shown recently that the transmembrane immunoadaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulates mouse liver DC maturation and proinflammatory and immune stimulatory functions. Here, we used PCR analysis and flow cytometry to characterize expression of DAP12 and its associated triggering receptor, triggering receptor expressed on myeloid cells 2 (TREM2), by mouse and human liver DCs and other immune cells compared with DCs in other tissues. We also examined the roles of DAP12 and TREM2 and their expression by liver DCs in the regulation of liver IRI. Injury was induced in DAP12-/- , TREM2-/- , or wild-type (WT) mice by 1 hour of 70% clamping and quantified following 6 hours of reperfusion. Both DAP12 and TREM2 were coexpressed at comparatively high levels by liver DCs. Mouse liver DCs lacking DAP12 or TREM2 displayed enhanced levels of nuclear factor κB and costimulatory molecule expression. Unlike normal WT liver DCs, DAP12-/- liver DC failed to inhibit proliferative responses of activated T cells. In vivo, DAP12-/- and TREM2-/- mice exhibited enhanced IRI accompanied by augmented liver DC activation. Elevated alanine aminotransferase levels and tissue injury were markedly reduced by infusion of WT but not DAP12-/- DC. Conclusion: Our data reveal a close association between DAP12 and TREM2 expression by liver DC and suggest that, by negatively regulating liver DC stimulatory function, DAP12 promotes their control of hepatic inflammatory responses; the DAP12/TREM2 signaling complex may represent a therapeutic target for control of acute liver injury/liver inflammatory disorders.

Ticagrelor Does Not Protect Isolated Rat Hearts, Thus Clouding Its Proposed Cardioprotective Role Through ENT 1 in Heart Tissue.

P2Y12 receptor-blocking drugs given at reperfusion offer protection against myocardial infarction in animal models of transient coronary occlusion. Two recent reports concluded that ticagrelor was more cardioprotective than clopidogrel and attributed this to ticagrelor's unique ability to raise tissue adenosine by blocking the equilibrative nucleoside transporter 1. Indeed, an adenosine receptor blocker attenuated ticagrelor's protection. The related P2Y12 inhibitor cangrelor, which does not block the transporter, protects hearts only when platelets are in the perfusate, while adenosine is known to protect equally in situ blood-perfused and crystalloid-perfused isolated hearts. We, therefore, tested whether ticagrelor liberates a sufficient amount of adenosine to protect a Krebs buffer-perfused isolated rat heart subjected to 40 minutes of global ischemia followed by 2 hours of reperfusion. In untreated hearts, 77.6% ± 4.0% of the ventricle was infarcted as measured by triphenyltetrazolium staining. Ischemically preconditioned hearts had only 32.7% ± 3.6% infarction ( P < .001 vs untreated), indicating that our model could be protected by preconditioning which is known to involve adenosine. Strikingly, hearts treated with 10 μmol/L ticagrelor in the buffer throughout the reperfusion period had 77.5% ± 2.4% infarction comparable to unprotected controls ( P = NS vs untreated). These data strongly suggest that ticagrelor was unable to release sufficient adenosine from the crystalloid-perfused rat heart to protect it against infarction. Our previous studies have found no difference in the anti-infarct potency among clopidogrel, cangrelor, and ticagrelor in open-chest rats and rabbits, and surprisingly adenosine receptor antagonists block protection from all 3 drugs. We have no explanation why ticagrelor is more protective in the pig than clopidogrel but suspect a species or perhaps a treatment schedule difference that may or may not involve adenosine.

Increase in post-reperfusion sensitivity to tissue plasminogen activator-mediated fibrinolysis during liver transplantation is associated with abnormal metabolic changes and increased blood product utilisation.

Increased systemic fibrinolytic activity can occur in liver transplant recipients after the donor graft is reperfused. However, it remains unclear whether this is related solely to tissue plasminogen activator (t-PA) levels or whether unique metabolic changes can alter t-PA activity and enhance fibrinolytic activity. We hypothesise that an increase in sensitivity to t-PA-mediated fibrinolysis (StF) following liver reperfusion is associated with specific metabolic abnormalities. Liver transplant recipients had serial blood samples analysed with a modified thrombelastography assay using exogenous t-PA to measure sensitivity/resistance to fibrinolysis with the lysis 30 min after maximum clot strength (tLY30). Paired plasma samples were analysed with mass spectroscopy-based metabolomics. The tLY30 was correlated to metabolites using Spearman's rho. StF was defined as a tLY30 change of >8.5% from the anhepatic phase to 30 min after reperfusion based on the distribution of tLY30 in a healthy control population. StF occurred in 53% of patients. Cohorts had similar MELD scores (18 vs 16, p=0.876) and tLY30 at baseline (p=0.867) and anhepatic phase of surgery (p=0.463). Thirty min after reperfusion, the tLY30 was 73% in patient with StF vs 33% in those without StF 33% (p=0.006). StF was associated with increased red blood cell transfusions (p=0.035), during the first 2 hours of reperfusion. Nine metabolites demonstrated a correlation with tLY30 (p<0.05). StF is a transient event that resolves within 2 hours of graft reperfusion and is associated with increased blood product use. This phenomenon correlates with derangements in citric acid cycle, purine and amino acid metabolism. Future research is needed to determine whether these metabolites are biomarkers or mechanistically linked to increased sensitivity to t-PA-mediated fibrinolytic activity following graft reperfusion.

Protective Effects of Galium verum L. Extract against Cardiac Ischemia/Reperfusion Injury in Spontaneously Hypertensive Rats.

Galium verum L. (G. verum, lady's bedstraw) is a perennial herbaceous plant, belonging to the Rubiaceae family. It has been widely used throughout history due to multiple therapeutic properties. However, the effects of this plant species on functional recovery of the heart after ischemia have still not been fully clarified. Therefore, the aim of our study was to examine the effects of methanol extract of G. verum on myocardial ischemia/reperfusion (I/R) injury in spontaneously hypertensive rats (SHR), with a special emphasis on the role of oxidative stress. Rats involved in the research were divided randomly into two groups: control (spontaneously hypertensive rats (SHR)) and G. verum group, including SHR rats treated with the G. verum extract (500 mg/kg body weight per os) for 4 weeks. At the end of the treatment, in vivo cardiac function was assessed by echocardiography. Rats were sacrificed and blood samples were taken for spectrophotometric determination of systemic redox state. Hearts from all rats were isolated and retrogradely perfused according to the Langendorff technique. After a stabilization period, hearts were subjected to 20-minute ischemia, followed by 30-minute reperfusion. Levels of prooxidants were spectrophotometrically measured in coronary venous effluent, while antioxidant enzymes activity was assessed in heart tissue. Cell morphology was evaluated by hematoxylin and eosin (HE) staining. 4-week treatment with G. verum extract alleviated left ventricular hypertrophy and considerably improved in vivo cardiac function. Furthermore, G. verum extract preserved cardiac contractility, systolic function, and coronary vasodilatory response after ischemia. Moreover, it alleviated I/R-induced structural damage of the heart. Additionally, G. verum extract led to a drop in the generation of most of the measured prooxidants, thus mitigating cardiac oxidative damage. Promising potential of G. verum in the present study may be a basis for further researches which would fully clarify the mechanisms through which this plant species triggers cardioprotection.

AP-1 (Activated Protein-1) Transcription Factor JunD Regulates Ischemia/Reperfusion Brain Damage via IL-1β (Interleukin-1β).

Background and Purpose- Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD-a member of the AP-1 (activated protein-1) family of transcription factors-was recently shown to regulate inflammation by targeting IL (interleukin)-1β synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods- WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1β was investigated by treating JunD siRNA mice with an anti-IL-1β monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results- In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1β levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti-IL-1β antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions- JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1β.

Abstract TP140: Intra-Arterial Mesenchymal Stem Cell Therapy Modulates Expression of NLRP1 Inflammasome in Animal Model of Ischemic Stroke

Background: Stroke is one of the leading causes of long-term disability worldwide. Ischemia, followed by acidosis, worsens the pathology by activating the acid sensing ion channel 1a (ASIC1a). This activated ASIC1a increases the expression of NLRP1 inflammasome in a pH dependent manner that further activates the inflammatory cascade. ASIC1a once activated triggers the influx of Ca 2+ ions and its overload contributes to neuronal injury and death in ischemic stroke. Cell based therapy with mesenchymal stem cells (MSCs) is promising for clinical translation and reported to improve neurological outcome in rodent model. We aim to test if intra-arterial (IA) MSCs can inhibit the production of inflammasome by attenuating ASIC1a expression to render neuroprotection. Methods: Middle-aged Sprague Dawley rodents were infused with IA MSCs at 6 hours post middle cerebral artery occlusion (MCAo) followed by 24 hours of reperfusion. Rats were evaluated for neurodeficit and motor functions. Brains were harvested for infarct size estimation, biochemical analysis and western blot experiments. Results: IA MSCs when given at a dose of 1x10 5 cells at 6 hours following ischemia confer neuroprotection as evident by reduction in infarct volume, improvement in neurodeficit scores and motor function. Increase in GSH levels and reduction in elevated nitrite and MDA levels were observed in the IA MSCs treated animals. IA MSCs infusion also attenuated the expression of ASIC1a, NLRP1, NLRP3, ASC1, IL1β and caspase-1 in the cortical brain regions following ischemia. Conclusion: IA MSCs confers neuroprotection by attenuating acidosis by modulating NLRP1 mediated ASIC1a expression. Keywords: Ischemic Stroke, Mesenchymal stem cell, Inflammasome, Interleukin-1β, Peri-infarct area

Abstract WMP83: cGMP-Dependent Protein Kinase I in Smooth Muscle Cells Protects Against Stroke in Female Mice

Introduction: The role of cGMP-dependent protein kinase I (cGKI) in regulating vascular smooth muscle cells (SMC) during stroke has not been completely clarified. We showed that SMC cGKI has protective effect against stroke in male mice. To elucidate whether cGKI in SMC renders its protective effect on stroke outcome by sex-dependent mechanism, we conducted study on female mice. Animals: We used a novel tamoxifen-inducible SMC-specific gene knockout mice, that expresses CreER T2 recombinase under the control of smooth muscle alpha-actin promoter. SMA-CreER T2 _cGKIfloxflox adult 12-14 weeks-old female mice after tamoxifen (TAM) administration (100 μl TAM solution (1 mg/ml) i.p. per day for 5 consecutive days at the age 5-6 weeks) were used as experimental animals (cGKI SMKO mice). Control animals consisted of three types of female mice: (1) C57BL/6 (WT) mice without TAM treatment; (2) SMA-CreER T2 _cGKIfloxflox mice without TAM treatment (cGKI control); (3) cGKIfloxflox mice (without SMA-CreER T2 transgene) treated with TAM (cGKI control TAM). Methods: Mice (n=12/group) were anesthetized (1.5% of isoflurane in 30% of O 2 and 70% of N 2 O), body temperature was maintained (36.5-37.0 0 C) during 30-min filament-mediated middle cerebral artery occlusion (MCAO) and 30 min of reperfusion. Cerebral laser-Doppler flowmetry was utilized during the surgery to evaluate the regional cerebral blood flow (CBF). Results: The cGKI SMKO mice revealed greater cerebral infarct volume (93±12 mm 3 , Mean±SD) than WT (64±14), cGKI control (62±14), and cGKI control TAM (68±10) mice (TTC staining, indirect calculation, p&lt;0.001, one-way analysis of variance with Bonferroni correction). Neurological deficit by 4-point scale was worse in the cGKI SMKO mice (2.3 points) as compared with WT (1.5), cGKI control (1.2), and cGKI control TAM (1.3) mice (p&lt;0.05). The CBF during reperfusion was less in cGKI SMKO mice (44±19%) than in control groups (70±16 in WT; 81±33 in cGKI control; 70±19 in cGKI control TAM (15-th minute of reperfusion), p&lt; 0.05). In conclusion, cGKI in SMC protects against stroke injury in female mice in a similar way that we previously detected in male mice. These results suggest that cGKI in SMC should be targeted in studies designed to protect brain tissue against stroke.

Abstract TP327: Plant Long-Chain Isoprenoid Alcohols (Polyprenols) Protect Against Cerebral Ischemia and Reperfusion Injury in a Mouse Model of Stroke

Introduction: Plant long-chain isoprenoid alcohols (polyprenols) are biologically active molecules affecting the isoprenoid pathway. Polyprenols have broad therapeutic actions including for infections and inflammatory, hepatic and neurological conditions. The aim of this study was to evaluate the protective effects of pharmaceutical-grade polyprenols on experimental stroke in a mouse middle cerebral artery occlusion (MCAO) followed by reperfusion model. Methods: Pharmaceutical-grade polyprenol substance, Ropren®, is isolated from green verdure of ( Picea abies (L) Karst) . Adult (12-14 weeks old) C57BL/6 male mice were anesthetized (1.5% of isoflurane in 30% of O 2 and 70% of N 2 O) and body temperature was maintained (36.5-37.0°C) during 30-min filament-mediated MCAO and 48 h of reperfusion. Cerebral laser-Doppler flowmetry was utilized during MCAO to test whether regional cerebral blood flow (CBF) differences correlated with stroke outcome. Before and 24 h after MCAO, the experimental group was treated with Ropren® intraperitoneal injections (12 mg/kg) diluted in 100 μl sunflower oil. The control group was treated with 100 μl oil only at the same time points. Mice were euthanized 48 h after MCAO and infarct volumes were calculated (indirect method) after TTC staining. Results: Experimental stroke revealed a decrease in the cerebral infarct volume in the Ropren-treated group (51±16 mm 3 , Mean±SD) in comparison with the control group (67±13) (n=10/group, p &lt; 0.05, two-tailed t-test). Neurological deficit measured by a 4-point scale was improved in Ropren-treated mice (1.2 points) compared with control mice (1.8) (n=10/group, p=0.05). We did not find a significant difference both between intra-ischemic CBF and CBF during reperfusion between groups (15th minute of reperfusion: 62±24% in Ropren-treated mice; 77±35% in control mice (n=10/group, NS)). This suggests Ropren® renders its neuroprotective effects by mechanisms independent of CBF. Conclusion: This study shows the protective effects of pharmaceutical-grade polyprenols (Ropren®) during cerebral ischemia-reperfusion injury in the mouse MCAO model. Further research is required to understand the mechanisms of Ropren-mediated neuroprotection in this ischemic stroke model.

Abstract TP459: Reducing Door-to Needle Time in Half-Lessons Learned

Background and Issues: For each minute reperfusion is delayed, 1.9 million neurons die. In early 2017, the door-to-needle (TPN) times for initiating Alteplase (TPA) for our institution were starting to creep up over fifty-six minutes. Though the Joint Commission’s minimum standard is to start TPA on eligible patients in less than sixty minutes on average, we wanted to do better. A team had utilized Six Sigma approaches in prior years and saw some improvement; however, in this effort the interprofessional team chose to utilize those methods as well creation of a culture shift toward patient-centered reduction of DTN time. Purpose: Reduce DTN time to an average of less than or equal to thirty minutes on eligible patients. Methods: The interprofessional team was established to represent key stakeholders who then reached consensus on the current process. Goal timeframes for the subcomponents of the process were determined. A fishbone diagram was used to identify key barriers to meeting time goals. The four barrier areas identified included: Scan interpretation; documentation and communication; location where TPA was administered; and completion of other tasks before TPA. Subgroups worked on each barrier category. A tracking feedback tool was revised and utilized for consistent feedback on each TPA case. The Get With the Guidelines criteria for sixty-minute TPA timeliness was used to determine eligible patients for the thirty-minute window. Information was shared with the team at least monthly. Recognition was provided to staff, providers, and emergency medical system units involved in fastest TPA administration times. Results: Quarterly DTN needle times dropped from 56.1 to 26.4 minutes for all patients and from 54.6 to 26.1 minutes for eligible patients (pre-task force/2nd quarter 2017 to final post-task force/2nd quarter 2018, respectively). The volume of patients receiving TPA also increased from 11.5 to 13.75 patients/quarter. Increased collegiality and joint problem solving between services and departments were also noted as a result of this effort. Conclusions: By emphasizing teamwork and collaboration, not only were DTN times dropped by more than half, the culture of care was enhanced to truly reflect the patient at the center.

Erythropoietin Ameliorates Lung Injury by Accelerating Pulmonary Endothelium Cell Proliferation via Janus Kinase-Signal Transducer and Activator of Transcription 3 Pathway After Kidney Ischemia and Reperfusion Injury

Background/aimsKidney ischemia and reperfusion injury could cause microvascular barrier dysfunction, lung inflammatory cascades activation, and programmed cell death of pulmonary endothelium, leading to acute lung injury. Our study aimed at determining whether erythropoietin (EPO) can ameliorate lung dysfunction following renal ischemia and reperfusion (IR) injury and explored the underlying mechanisms. MethodsIn vivo, C57BL/6 mice received EPO (6000 U/kg) before right renal vascular pedicles clamping for 30 minutes, followed by 24 hours of reperfusion. The lung histopathologic changes and inflammatory cytokines expression were assessed. In vitro, cultured human umbilical vein endothelial cells were treated with EPO, and apoptosis rate, proliferation capacity, and phosphorylation status of the Janus kinase-signal transducer and activator of transcription 3 (Jak-STAT3) pathway were measured respectively in the presence or absence of lipopolysaccharide stimulation. ResultsIn vivo, EPO remarkably attenuated pulmonary interstitial and alveolar epithelial edema caused by renal IR injury. In vitro, the proliferation capacity of human umbilical vein endothelial cells was significantly increased under EPO stimulation, which correlated with changes in Jak-STAT3 signaling. ConclusionOur data indicated that EPO is able to ameliorate acute lung tissue damage induced by renal IR, and at least in part, via the Jak-STAT3 pathway.

Rapid Alterations in Cerebral White Matter Lipid Profiles After Ischemic-Reperfusion Brain Injury in Fetal Sheep as Demonstrated by MALDI-Mass Spectrometry.

Perinatal ischemia-reperfusion (I/R) injury of cerebral white matter causes long-term cognitive and motor disabilities in children. I/R damages or kills highly metabolic immature oligodendroglia via oxidative stress, excitotoxicity, inflammation, and mitochondrial dysfunction, impairing their capacity to generate and maintain mature myelin. However, the consequences of I/R on myelin lipid composition have not been characterized. This study utilized matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to assess alterations in cerebral supraventricular white matter myelin lipid profiles in a fetal sheep model of perinatal I/R. Fetal sheep (127 days gestation) were studied after 30 minutes of bilateral carotid artery occlusion followed by 4 (n = 5), 24 (n = 7), 48 (n = 3), or 72 (n = 5) hours of reperfusion, or sham treatment (n = 5). White matter lipids were analyzed by negative ion mode MALDI-MS. Striking I/R-associated shifts in phospholipid and sphingolipid expression occurred over the 72-hour time course with most responses detected within 4 hours of reperfusion and progressing at the 48- and 72-hour points. I/R decreased expression of phosphatidic acid and phosphatidylethanol amine and increased phosphatidylinositol, sulfatide, and lactosylceramide. Cerebral I/R in mid-gestation fetal sheep causes rapid shifts in white matter myelin lipid composition that may reflect injury, proliferation, or recovery of immature oligodendroglia.

An optimized low-pressure tourniquet murine hind limb ischemia reperfusion model: Inducing acute ischemia reperfusion injury in C57BL/6 wild type mice.

Acute ischemia reperfusion injury in skeletal muscle remains an important issue in several fields of regenerative medicine. Thus, a valid model is essential to gain deeper insights into pathophysiological relations and evaluate possible treatment options. While the vascular anatomy of mice regularly prevents sufficient vessel occlusion by invasive methods, there is a multitude of existing models to induce ischemia reperfusion injury without surgical procedures. Since there is no consensus on which model to prefer, this study aims to develop and evaluate a novel, optimized low-pressure tourniquet model. C57BL/6 mice underwent an ischemic procedure by either tourniquet or invasive artery clamping. A sham group served as control. With exception of the sham group, mice underwent 2 hours of ischemia followed by 4 hours of reperfusion. Groups were compared using microcirculatory and spectroscopic measurements, distinctions in tissue edema, histological and immunohistochemical analyses. Both procedures led to a significant decrease in tissue blood flow (- 97% vs. - 86%) and oxygenation (- 87% vs. - 75%) with a superiority of the low-pressure tourniquet. Tissue edema in the tourniquet cohort was significantly increased (+ 59%), while the increase in the clamping cohort was non-significant (+ 7%). Haematoxylin Eosin staining showed significantly more impaired muscle fibers in the tourniquet group (+ 77 p.p. vs. + 11 p.p.) and increased neutrophil infiltration/ROI (+ 51 vs. + 8). Immunofluorescence demonstrated an equal increase of p38 in both groups (7-fold vs. 8-fold), while the increase in apoptotic markers (Caspase-3, 3-Nitrotyrosine, 4-Hydroxynonenal) was significantly higher in the tourniquet group. The low-pressure tourniquet has been proven to produce reproducible and thus reliable ischemia reperfusion injury. In addition, significantly less force was needed than previously stated. It is therefore an important instrument for studying the pathophysiology of ischemia reperfusion injury and for the development of prophylactic as well as therapeutic interventions.