Transient Ligation Research Articles

Role of inflammatory populations and their intermediates in post-ischemic adverse remodeling in rat model of myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía- Fondos FEDER Background Compelling evidence demonstrated the critical role of inflammation in ventricular adverse remodeling after acute myocardial infarction. However, it remains unclear how inflammatory cell population and their intermediates participate in the progress of the disease. Purpose This study aims to elucidate the role of inflammatory population in cardiac remodeling after myocardial infarction in a rat model, focusing on their role in fibrosis. Methods We used a surgical procedure by transient ligation of the left descendant coronary artery, to evoke ischemia-reperfusion (I/R) in rats. We performed FACS analysis to evaluate counts of neutrophils and two monocytes’ subsets: CD43lowHis48high (classical monocytes), and CD43highHis48low (non-classical monocytes), which are the most pro-inflammatory subset in peripheral blood. Subsequently, we performed immunofluorescence to detect pro-inflammatory M1 and M2 macrophages (CD68+ and CD163+) in the rat infarcted myocardium. Moreover, we sorted the two monocyte subsets and performed genes and microRNAs Array. Results FACS analysis of neutrophils and monocytes subsets showed an increased level of these cells in the rat model of myocardial infarction, one week after the post-ischemic event. In parallel, the infiltration of M1 macrophages was increased in the myocardium until 1 week after surgery, indicating a long extravasation of inflammatory cells after the ischemic event. Moreover, co-culture of fibroblasts and cardiomyocytes with intermediates released by inflammatory cells subjected to I/R increased the expression of fibrotic and apoptotic genes. In addition, the analysis of genes’ Array by GSEA indicated that CD43highHis48low monocytes had an enrichment score in pathways associated with angiogenesis, fibrosis and inflammation, while CD43lowHis48high population exhibited an enrichment in cytokines and inflammation, apoptosis and fibrosis. Finally, we found significant overexpression of microRNAs, for instance, miR-27a-3p, miR-29b-3p, miR-30e-5p and miR-194-5p in CD43highHis48low 24h after the I/R procedure. Those miRNAs are predicted to modulate pro-fibrotic genes. Conclusions The level of inflammatory cell populations is increased in I/R rat model. Moreover, these cells infiltrate in the myocardium likely to change the transcriptome of resident cells towards a pro-apoptotic and pro-fibrotic phenotype.

B cell subsets contribute to myocardial protection by inducing neutrophil apoptosis after ischemia and reperfusion.

A robust, sterile inflammation underlies myocardial ischemia and reperfusion injury (MIRI). Several subsets of B cells possess the immunoregulatory capacity that limits tissue damage, yet the role of B cells in MIRI remains elusive. Here, we sought to elucidate the contribution of B cells to MIRI by transient ligation of the left anterior descending coronary artery in B cell-depleted or -deficient mice. Following ischemia and reperfusion (I/R), regulatory B cells are rapidly recruited to the heart. B cell-depleted or -deficient mice exhibited exacerbated tissue damage, adverse cardiac remodeling, and an augmented inflammatory response after I/R. Rescue and chimeric experiments indicated that the cardioprotective effect of B cells was not solely dependent on IL-10. Coculture experiments demonstrated that B cells induced neutrophil apoptosis through contact-dependent interactions, subsequently promoting reparative macrophage polarization by facilitating the phagocytosis of neutrophils by macrophages. The in vivo cardioprotective effect of B cells was undetectable in the absence of neutrophils after I/R. Mechanistically, ligand-receptor imputation identified FCER2A as a potential mediator of interactions between B cells and neutrophils. Blocking FCER2A on B cells resulted in a reduction in the percentage of apoptotic neutrophils, contributing to the deterioration of cardiac remodeling. Our findings unveil a potential cardioprotective role of B cells in MIRI through mechanisms involving FCER2A, neutrophils, and macrophages.

Growth differentiation factor 15 (GDF15) preconditioning but not post-conditioning protects the hearts towards ischemia-reperfusion injury

Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine, which can be produced under certain pathological situations, mainly related to inflammatory stress, aging and disease. While clinical data suggest that GDF15 is a powerful prognosis factor in several high-risk cardiovascular situations, experimental studies rather consider this cytokine as a cardioprotective molecule. We aimed to clarify the direct cardiac effects of GDF15 during ischemia-reperfusion (I-R) injury, at concentrations found in patients with high cardiovascular risk, such as these of patients presenting with acute myocardial infarction or stroke. Wistar male rats (200–400 g) were used for both in vivo and ex vivo ischemia-reperfusion and anesthetized with isoflurane. In vivo rats were injected with either saline (Control groups) or 2.5 μg/kg of GDF15 (GDF15 groups), 20 minutes before ischemia in the preconditioning protocol, or at the end of ischemia in the post-conditioning protocol. Myocardial ischemia was induced by the transient ligation of the left anterior descending artery for 30 min followed by 24 h of reperfusion. In a second set of experiments, rat's hearts were isolated and perfused ex vivo on a Langendorff model with either saline (Control groups) or 2000 ng/L of GDF15 upstream the coronary bed (GDF15 groups) for 10 minutes before ischemia or during reperfusion following 30 minutes of global normothermic total ischemia. GDF15 decreased the infarct size in vivo as well as in ex vivo when administrated before ischemia but not at the end of it. Moreover, in our ex vivo model, only GDF15 preconditioning induced a better recovery of most contractile parameters after ischemia (P < 0.05 for all parameters). These results demonstrate that exogenous GDF15 preconditioning decreased cardiac cell death in vivo but also ex vivo, the latter situation involving that inflammatory, endocrine, and nervous systems are not interfering. Suggesting that GDF15 exerts direct cardioprotective properties towards ischemia-reperfusion injury.

Characterization of early myocardial inflammation in ischemia-reperfusion injury.

Myocardial injury may be caused by myocardial ischemia-reperfusion (IR), and salvaging such an injury is still a great challenge in clinical practice. This study comprehensively characterized the physiopathologic changes of myocardial injury after IR to explore the underlying mechanism in the early reperfusion phase with particular emphasis on early myocardial inflammation. The experimental IR model was obtained by the left anterior descending artery's transient ligation of C57BL/6 mice. T2W signals of all mice showed increased signal at different IR stages. It was positively correlated with inflammatory cytokines and cells. T2W imaging by 7.0 T MRI surprisingly detected signal enhancement, but histopathology and flow cytometry did not reveal any inflammatory cells infiltration within 3 h after IR. Cardiomyocyte swelling and increased vascular permeability were observed by WGA staining and ultrastructural analysis, respectively. The 3 h IR group showed that the cardiomyocytes were severely affected with disintegrating myofilaments and mitochondria. Both VEGF and phosphorylated Src protein were markedly expressed in the 3 h IR group in comparison with the sham group, and TUNEL staining displayed little positive cells. Cleaved caspase-3 apoptin also has similar expression levels with that of the sham group. Resident macrophages had notably become M1 phenotype. The T2W signal was still elevated, and we observed that collagen deposition occurred from 1 to 7 days. The inflammation response during the first week after reperfusion injury gradually increase 3 h later, but the main manifestation before that was edema. This study indicated that the first 3 h may be crucial to the early rescue process for reperfusion-induced myocardial injury due to inflammatory cell infiltration absence and apoptosis.

Detection of apoptosis by [18F]ML-10 after cardiac ischemia–reperfusion injury in mice

ObjectiveMyocardial infarction leads to ischemic heart disease and cell death, which is still a major obstacle in western society. In vivo imaging of apoptosis, a defined cascade of cell death, could identify myocardial tissue at risk.MethodsUsing 2-(5-[18F]fluoropentyl)-2-methyl-malonic acid ([18F]ML-10) in autoradiography and positron emission tomography (PET) visualized apoptosis in a mouse model of transient ligation of the left anterior descending (LAD) artery. 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) PET imaging indicated the defect area. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) histology stain indicated cardiac apoptosis.Results[18F]ML-10 uptake was evident in the ischemic area after transient LAD ligation in ex vivo autoradiography and in vivo PET imaging. Detection of [18F]ML-10 is in line with the defect visualized by [18F]FDG and the histological approach of TUNEL staining.ConclusionThe tracer [18F]ML-10 is suitable for detecting apoptosis after transient LAD ligation in mice.

Exogenous growth differentiation factor 15 (GDF15) exerts direct cardioprotective properties towards myocardial ischemia reperfusion injury

Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine which can be produced under certain pathological situations, mainly related to inflammatory stress, aging and disease. While clinical data suggest that GDF15 is a powerful risk factor in several high-risk cardiovascular situations such as myocardial infarction and coronary artery disease, experimental studies rather consider this cytokine as a cardioprotective molecule. We aimed to clarify the direct cardiac effects of GDF15 during ischemia-reperfusion (I-R) injury, at concentrations found in patients with high cardiovascular risk, such as these of patients presenting with acute myocardial infarction or stroke. Wistar male rats (200–400 g) were used for both in vivo and ex vivo ischemia-reperfusion and anesthetized with isoflurane. In vivo rats were intubated before a left thoracotomy was performed between the 3rd and the 4th intercostal space. Rats were injected with either saline (Control groups) or 2.5 μg/kg of GDF15 (GDF15 groups), 20 minutes before the transient ligation of the left anterior descending artery for 30 min followed by 24 h of reperfusion. Ex vivo rat's hearts were isolated and perfused on a Langendorff model with either saline (Control groups) or 2000 ng/L of GDF15 upstream the coronary bed (GDF15 groups) for 10 minutes, then subjected to either 20 or 30 minutes of global normothermic total ischemia, followed by 2 h of reperfusion. GDF15 decreased the infarct size in vivo as well as in ex vivo. Moreover, in our ex vivo model, GDF15 induced a better recovery of most contractile parameters after ischemia ( P < 0.05 for all parameters). These results demonstrate for the first time that exogenous preischemic short-term administration of GDF15 decreased cardiac cell death in vivo but also ex vivo, the latter situation involving that inflammatory, endocrine, and nervous systems are not interfering. These original results suggest that GDF15 exerts direct cardioprotective properties towards ischemia-reperfusion injury.

Comparison of transient and permanent LAD ligation in mice using 18F-FDG PET imaging

ObjectiveAnimal models for myocardial injuries represent important cornerstones in cardiovascular research to monitor the pathological processes and therapeutic approaches. We investigated the association of 18F-FDG derived left ventricular metabolic volume (LVMV), defect area and cardiac function in mice after permanent or transient ligation of the left anterior descending artery (LAD).MethodsSerial non-invasive ECG-gated 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography (18F-FDG PET) after permanent or transient LAD ligation enabled a longitudinal in vivo correlation of 18F-FDG derived left ventricular metabolic volume to functional parameters and myocardial defect.ResultsThe LVMV shows a more prominent drop after permanent than transient LAD ligation and recovers after 30 days. The loss of LVMV correlates with the defect area assessed by QPS software. Cardiac function parameters (e.g., EDV, ESV, SV) by the QGS software positively correlate with LVMV after permanent and transient LAD ligation.ConclusionsThis study provides novel insight into 18F-FDG derived LVMV after permanent and transient LAD ligation by longitudinal in 18F-FDG PET imaging and underlines the associations of the FDG derived parameter and cardiac function.

CCR6 Deficiency Increases Infarct Size after Murine Acute Myocardial Infarction.

Ischemia-reperfusion injury after the reopening of an occluded coronary artery is a major cause of cardiac damage and inflammation after acute myocardial infarction. The chemokine axis CCL20-CCR6 is a key player in various inflammatory processes, including atherosclerosis; however, its role in ischemia-reperfusion injury has remained elusive. Therefore, to gain more insight into the role of the CCR6 in acute myocardial infarction, we have studied cardiac injury after transient ligation of the left anterior descending coronary artery followed by reperfusion in Ccr6−/− mice and their respective C57Bl/6 wild-type controls. Surprisingly, Ccr6−/− mice demonstrated significantly reduced cardiac function and increased infarct sizes after ischemia/reperfusion. This coincided with a significant increase in cardiac inflammation, characterized by an accumulation of neutrophils and inflammatory macrophage accumulation. Chimeras with a bone marrow deficiency of CCR6 mirrored this adverse Ccr6−/− phenotype, while cardiac injury was unchanged in chimeras with stromal CCR6 deficiency. This study demonstrates that CCR6-dependent (bone marrow) cells exert a protective role in myocardial infarction and subsequent ischemia-reperfusion injury, supporting the notion that augmenting CCR6-dependent immune mechanisms represents an interesting therapeutic target.

Hexarelin targets neuroinflammatory pathways to preserve cardiac morphology and function in a mouse model of myocardial ischemia-reperfusion

Acute myocardial ischemia and reperfusion injury (IRI) underly the detrimental effects of coronary heart disease on the myocardium. Despite the ongoing advances in reperfusion therapies, there remains a lack of effective therapeutic strategies for preventing IRI. Growth hormone secretagogues (GHS) have been demonstrated to improve cardiac function, attenuate inflammation and modulate the autonomic nervous system (ANS) in models of cardiovascular disease. Recently, we demonstrated a reduction in infarct size after administration of hexarelin (HEX), in a murine model of myocardial infarction. In the present study we employed a reperfused ischemic (IR) model, to determine whether HEX would continue to have a cardioprotective influence in a model of higher clinical relevance.Myocardial ischemia was induced by transient ligation of the left descending coronary artery (tLAD) in C57BL/6 J mice followed by HEX (0.3 mg/kg/day; n = 20) or vehicle (VEH) (n = 18) administration for 21 days, first administered immediately prior-to reperfusion. IR-injured and sham mice were subjected to high-field magnetic resonance imaging to assess left ventricular (LV) function, with HEX-treated mice demonstrating a significant improvement in LV function compared with VEH-treated mice. A significant decrease in interstitial collagen, TGF-β1 expression and myofibroblast differentiation was also seen in the HEX-treated mice after 21 days. HEX treatment shifted the ANS balance towards a parasympathetic predominance; combined with a significant decrease in cardiac troponin-I and TNF-α levels, these findings were suggestive of an anti-inflammatory action on the myocardium mediated via HEX.In this model of IR, HEX appeared to rebalance the deregulated ANS and activate vagal anti-inflammatory pathways to prevent adverse remodelling and LV dysfunction. There are limited interventions focusing on IRI that have been successful in improving clinical outcome in acute myocardial infarction (AMI) patients, this study provides compelling evidence towards the translational potential of HEX where all others have largely failed.

Mesenchymal Stem Cells Promote the Resolution of Cardiac Inflammation After Ischemia Reperfusion Via Enhancing Efferocytosis of Neutrophils.

BackgroundNeutrophils play a major role in inflammation after myocardial ischemia‐reperfusion (I/R) injury. The effects of mesenchymal stem cells (MSCs) on neutrophils in I/R are complex and not fully understood. This study was designed to investigate the effects and mechanism of MSCs on alleviating myocardial I/R injury in rats.Methods and ResultsMSCs induced M2 macrophages polarization in vitro and enhanced macrophage efferocytosis of apoptotic neutrophils, measured by fluorescence‐activated cell sorting analysis and immunofluorescence staining. Rats myocardial I/R were induced by transient ligation of left anterior descending coronary. Adipose‐derived MSCs or vehicle were infused at initiation (immediate after reperfusion) or peak of inflammation (24 hours after I/R). Hematoxylin and eosin, 2,3,5‐triphenyltetrazolium chloride/Evans Blue staining and immunofluorescence staining were applied within 72 hours after cell infusion. Cardiac function was assessed by echocardiography and left cardiac catheterization analysis at 28 days post‐operation. MSCs infused immediately and 24 hours later both markedly ameliorated myocardial I/R injury, and immediate infusion had more significant outcome. These improvements were associated with neutrophils infiltration, measured by fluorescence‐activated cell sorting analysis and immunofluorescence staining. When infused immediately, MSCs did not significantly change neutrophil number at 24 hours but CD11b expression was significantly higher. When infused at 24 hours, MSCs markedly decreased neutrophil number by enhanced M2 macrophage infiltration and macrophage efferocytosis of neutrophils within 72 hours.ConclusionsEfferocytosis is pivotal to relieve neutrophil‐mediated I/R injury and initial the immune response for healing. MSCs infusion improves cardiac function in rats after myocardial I/R via the possible mechanism of enhancing M2 macrophages‐induced efferocytosis of apoptotic neutrophils.

The NLRP3 Inflammasome Inhibitor, OLT1177 (Dapansutrile), Reduces Infarct Size and Preserves Contractile Function After Ischemia Reperfusion Injury in the Mouse.

Activation of the NLRP3 inflammasome is a primary driver of sterile inflammation in response to myocardial ischemia reperfusion. Pharmacologic inhibitors of the NLRP3 inflammasome are being developed. We proposed that OLT1177 (dapansutrile), a novel NLRP3 inflammasome inhibitor, could preserve myocardial function after ischemia reperfusion injury in the mouse. We used an experimental murine model of myocardial ischemia reperfusion injury through transient ligation of the left coronary artery and measured the effects of OLT1177 (6, 60, or 600 mg/kg intraperitoneal dose) on infarct size at pathology and on systolic cardiac function at echocardiography. To simulate a clinical scenario, we investigated the time window of therapeutic intervention with OLT1177 (60 mg/kg) administered 60, 120, or 180 minutes after reperfusion. OLT1177 was rapidly detectable in the plasma following intraperitoneal injection and had no effect on cardiac function in healthy mice. OLT1177 treatment at reperfusion showed significant dose-dependent reduction in infarct size (-36%, -67%, and -62% for 6, 60, and 600 mg/kg, respectively; P < 0.001 for linear trend, P = 0.010 vs. vehicle for 6 mg/kg, and P < 0.001 vs. vehicle for 60 and 600 mg/kg) and preserved cardiac systolic function measured as left ventricular fractional shortening at 24 hours and 7 days after injury (P = 0.015 for 6 mg/kg and P < 0.01 for 60 and 600 mg/kg). OLT1177 reduced infarct size also when given after 60 minutes of reperfusion (-71%, P < 0.001 vs. vehicle). OLT1177 (dapansutrile) limits infarct size and prevents left ventricular systolic dysfunction when given within 60 minutes following ischemia reperfusion injury in the mouse.

Evaluation of retinal injury in a rat model of transient ischemic stroke.

Stroke-associated ocular disorders are vision-threatening. This study was designed to evaluate in vivo retinal injury induced by transient global cerebral ischemia/reperfusion (I/R). A stroke-induced retinal injury model in Wistar rats was established by electrocoagulation of bilateral vertebral arteries, combined with transient ligation of the bilateral common carotid arteries. Rats were randomly divided into groups based on the time post cerebral perfusion (3 h, 24 h, 48 h, 72 h, and 7 days). Retinal injury was evaluated by histological analysis, examination of eye fundus, and TUNEL staining. The expression of protein kinase C-alpha (PKCα) and fibrillary acidic protein (GFAP) was determined using qRT-PCR and immunofluorescence analysis. Both retinal neurons and the vasculature underwent significant damage in the cerebral-I/R groups when compared to rats in the sham group. Moreover, when compared to non-stroke rats, TUNEL staining revealed signs of apoptosis in the retina after transient ischemic stroke was induced (P<0.001). In these rats, the expression of PKCα and GFAP in the retinas was enhanced and peaked at 72 h after induction of cerebral-I/R (P<0.001). In this study, we found that retinas are very susceptible to transient global cerebral-I/R injury. The expression of PKCα and GFAP may be implicated in the pathogenesis of ischemic stroke-induced retinal injury.

Hypoxia/reperfusion predisposes to atherosclerosis.

Surgical interventions on blood vessels bear a risk for intimal hyperplasia and atherosclerosis as a consequence of injury. A specific feature of intimal hyperplasia is the loss of vascular smooth muscle cell (VSMC) differentiation gene expression. We hypothesized that immediate responses following injury induce vascular remodeling. To differentiate injury due to trauma, reperfusion and pressure changes we analyzed vascular responses to carotid artery bypass grafting in mice compared to transient ligation. As a control, the carotid artery was surgically laid open only. In both, bypass or ligation models, the inflammatory responses were transient, peaking after 6h, whereas the loss of VSMC differentiation gene expression persisted. Extended time kinetics showed that transient carotid artery ligation was sufficient to induce a persistent VSMC phenotype change throughout 28 days. Transient arterial ligation in ApoE knockout mice resulted in atherosclerosis in the transiently ligated vascular segment but not on the not-ligated contralateral side. The VSMC phenotype change could not be prevented by anti-TNF antibodies, Sorafenib, Cytosporone B or N-acetylcysteine treatment. Surgical interventions involving hypoxia/reperfusion are sufficient to induce VSMC phenotype changes and vascular remodeling. In situations of a perturbed lipid metabolism this bears the risk to precipitate atherosclerosis.

Complete remission of advanced hepatocellular carcinoma following transient chemoembolization and portal vein ligation

BackgroundMacroscopic diffuse-type hepatocellular carcinoma with concomitant major portal vein tumor thrombus (PVTT) and peritoneal dissemination indicates poor prognosis. Additionally, triple-positive tumor marker status is a predictor of poor outcome even after hepatectomy. Sorafenib is recommended in such patients, but it has limited therapeutic effectiveness.Case presentationA 54-year-old man was diagnosed with a liver abscess that was treated by puncture and drainage at a regional hospital. However, the diagnosis was subsequently changed to hepatocellular carcinoma with macroscopic portal vein tumor thrombus, based on the results obtained for the triple-positive tumor markers (alpha-fetoprotein, 45,928 ng/ml; protein induced by vitamin K absence or antagonist-II, 125,350 mAU/ml; and alpha-fetoprotein-L3, 38.3%). As the patient’s liver functional reserve was not adequate for curative resection, chemoembolization was performed with a hepatic arterial infusion of cisplatin (50 mg) and 5-FU (1000 mg), followed by mild embolization with cisplatin (50 mg) suspended in lipiodol (5 ml) and starch microspheres (300 mg) containing mitomycin C (4 mg). As the thrombus had progressed to the bifurcation of the right and left portal veins, the right vein was surgically ligated. Three peritoneal nodules could be identified and were removed. Three additional rounds of hepatic arterial chemotherapy/chemoembolization were performed after the initial surgery. At the 2-year evaluation, all tumor markers were observed to have normalized and diagnostic imaging showed complete remission.ConclusionsComplete remission of hepatocellular carcinoma with macroscopic portal vein tumor thrombus and peritoneal dissemination was obtained with a treatment regimen that involved four rounds of hepatic arterial infusion chemotherapy and transient chemoembolization, portal vein ligation, and the removal of peritoneal dissemination. This regimen can be recommended for patients with advanced hemiliver lesions who cannot undergo curative resection.

Urocortin-2 Prevents Dysregulation of Ca2+ Homeostasis and Improves Early Cardiac Remodeling After Ischemia and Reperfusion.

Aims: Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. However, little is known about its role in the regulation of intracellular Ca2+ concentration ([Ca2+]i) under I/R. Here, we examined whether the addition of Ucn-2 in reperfusion promotes cardioprotection focusing on ([Ca2+]i handling.Methods and Results: Cardiac Wistar rat model of I/R was induced by transient ligation of the left coronary artery and experiments were conducted 1 week after surgery in tissue and adult cardiomyocytes isolated from risk and remote zones. We observed that I/R promoted significant alteration in cardiac contractility as well as an increase in hypertrophy and fibrosis in both zones. The study of confocal [Ca2+]i imaging in adult cardiomyocytes revealed that I/R decreased the amplitude of [Ca2+]i transient and cardiomyocytes contraction in risk and remote zones. Interestingly, intravenous infusion of Ucn-2 before heart’s reperfusion recovered significantly cardiac contractility and prevented fibrosis, but it didn’t affect cardiac hypertrophy. Moreover, Ucn-2 recovered the amplitude of [Ca2+]i transient and modulated the expression of several proteins related to [Ca2+]i homeostasis, such as TRPC5 and Orai1 channels. Using Neonatal Rat Ventricular Myocytes (NRVM) we demonstrated that Ucn-2 blunted I/R-induced Store Operated Ca2+ Entry (SOCE), decreased the expression of TRPC5 and Orai1 as well as their interaction in reperfusion.Conclusion: Our study provides the first evidences demonstrating that Ucn-2 addition at the onset of reperfusion attenuates I/R-induced adverse cardiac remodeling, involving the [Ca2+]i handling and inhibiting the expression and interaction between TRPC5 and Orai1.

Monitoring of Myocutaneous Flaps by Measuring Capillary Glucose and Lactate Levels: Experimental Study.

In surgery, certain defects require reconstruction with a microsurgical flap. The free flap failure rate varies between 2% and 5%. Vascular thrombosis is the most frequent complication and represents 15% to 73% of failures. The success rate of salvage therapy is greater when salvage surgery is early. Currently, clinical monitoring is the criterion standard but many noninvasive or minimally invasive techniques have been developed to improve early diagnosis of complications of vascular thrombosis. The aim of our experimental study was to compare clinical assessments with measurements of capillary glycemia and lactatemia during the monitoring of free flaps. Myocutaneous latissimus dorsi flaps with skin paddles were created in pigs under general anesthesia. For each animal, 2 flaps were created (right and left) using the same technique. Four groups were made: group 1 (no flap ligation: control group), group 2 (flap with permanent ligation of the artery), group 3 (flap with permanent ligation of the two veins), group 4 (flap with transient ligation of the artery and 2 veins for 1 hour). The postoperative monitoring protocol consisted of monitoring the clinical, biological (glucose and lactate), and histological parameters. Eight animals were operated on and sixteen flaps were created. Each flap was clinically and biologically tested 25 times. Clinical, biological, and histological monitoring showed significant variations between the groups. The analysis of variance of capillary glycemia and lactatemia showed statistically significant difference between control group and group 2 (P < 0,0001), group 3 (P < 0,0001), or group 4 (P < 0,0001). There were no histological abnormalities after transient ligature at different times contrary to permanent ligature. Measuring capillary levels of lactate and glucose associated with clinical monitoring may shorten the time to diagnosis of flap failure. Ultimately, this will save lives and achieve better functional and aesthetic results.

Sustained Placental Growth Factor-2 Treatment Does Not Aggravate Advanced Atherosclerosis in Ischemic Cardiomyopathy.

Angiogenic growth factor therapy for ischemic cardiovascular disease carries a risk of stimulating atherosclerotic plaque growth. We evaluated risk benefit ratio of sustained administration of recombinant human placental growth factor (rhPlGF)-2 in mice with advanced atherosclerosis and chronic ischemic cardiomyopathy. We maintained apolipoprotein E-deficient mice on a high cholesterol diet and induced myocardial infarction by transient ligation at 4weeks. At 8weeks, we assessed left ventricular (LV) function and randomized mice to receive rhPlGF-2 or vehicle (VEH) subcutaneously for 28days. Administration of rhPlGF-2 significantly increased PlGF plasma levels without adverse hemodynamic or systemic inflammatory effects. RhPlGF-2 did not increase plaque area, composition, or vulnerability in the aortic arch. RhPlGF-2 significantly improved contractile function and reduced LV end-systolic and end-diastolic volume indices with a concomitant increase in capillary and arteriolar density in ischemic myocardium. RhPlGF-2 may represent a promising therapeutic strategy in chronic ischemic cardiomyopathy.

Glaucocalyxin A Ameliorates Myocardial Ischemia-Reperfusion Injury in Mice by Suppression of Microvascular Thrombosis.

BackgroundThe aim of this study was to evaluate the cardio-protective roles of glaucocalyxin A (GLA) in myocardial ischemia-reperfusion injury and to explore the underlying mechanism.Material/MethodsMyocardial ischemia-reperfusion in wild-type C57BL/6J mice was induced by transient ligation of the left anterior descending artery. GLA or vehicle (solvent) was administrated intraperitoneally to the mice before reperfusion started. After 24 h of myocardial reperfusion, ischemic size was revealed by Evans blue/TTC staining. Cardiac function was evaluated by echocardiography and microvascular thrombosis was assessed by immunofluorescence staining of affected heart tissue. We also measured the phosphorylation of AKT, ERK, P-GSK-3β, and cleaved caspase 3 in the myocardium.ResultsCompared to the solvent-treated control group, GLA administration significantly reduced infarct size (GLA 13.85±2.08% vs. Control 18.95±0.97%, p<0.05) and improved left ventricular ejection fraction (LVEF) (GLA 53.13±1.11% vs. Control 49.99±1.25%, p<0.05) and left ventricular fractional shortening (LVFS) (28.34±0.71% vs. Control 25.11±0.74%, p<0.05) in mice subjected to myocardial ischemia-reperfusion. GLA also attenuated microvascular thrombosis (P<0.05) and increased the phosphorylation of pro-survival kinase AKT (P<0.05) and GSK-3β (P<0.05) in the myocardium upon reperfusion injury.ConclusionsAdministration of GLA before reperfusion ameliorates myocardial ischemia-reperfusion injury in mice. The cardio-protective roles of GLA may be mediated through the attenuation of microvascular thrombosis.

Inhibition of the NLRP3 inflammasome limits the inflammatory injury following myocardial ischemia–reperfusion in the mouse

BackgroundSuccessful reperfusion is the most effective strategy to reduce ischemic injury in acute myocardial infarction (AMI). Ischemic injury, however, also triggers a secondary ischemia–independent injury, known as reperfusion injury, contributing to the overall infarct size. We hypothesize that inhibition of the Nod-like Receptor Protein-3 (NLRP3) inflammasome limits infarct size following myocardial ischemia/reperfusion (I/R), by inhibiting the inflammatory component of the reperfusion injury. MethodsCD-1 male mice underwent transient ligation of the left anterior descending coronary artery for 30 or 75min followed by reperfusion. Infarct size was measured at 1, 3 and 24h. A NLRP3 inflammasome inhibitor (NLRP3inh) or vehicle was administrated immediately at time of reperfusion or with a delay of 1 or 3h of reperfusion. ResultsA time-dependent increase in infarct size was measured at 1, 3, and 24h after reperfusion (11±2%, 30±5% and 43±4% of the area at risk respectively; P<0.001 for trend). NLRP3 myocardial expression was significantly increased at 24h and 6h vs 3h (P<0.01). Administration of the NLRP3inh at reperfusion did not reduce infarct size at 3h, while it significantly reduced infarct size at 24h (−56% vs vehicle, P<0.01). The NLRP3inh given 1h after reperfusion also significantly decreased caspase-1 activity and infarct size measured at 24h, whereas the NLRP3inh did not when given with a delay of 3h. ConclusionsPharmacological inhibition of the NLRP3 inflammasome within 1h of reperfusion limits the secondary inflammatory injury and infarct size following myocardial ischemia–reperfusion in the mouse.

Abstract 15506: Inhibition of the NLRP3 Inflammasome Limits the Inflammatory Injury Following Myocardial Ischemia-reperfusion in the Mouse

Introduction: Acute myocardial infarction (AMI) initiates as a result of severe ischemia, and while reperfusion relieves ischemic injury, limiting the overall infarct size (IS), it also triggers a secondary ischemia-independent myocardial injury occurs contributing to the final IS. Hypothesis: We hypothesize that inhibition of the Nod-like Receptor Protein-3 (NLRP3) inflammasome limits the progression of IS following myocardial ischemia/reperfusion (I/R). Methods: Adult CD1 male mice were subjected to transient ligation the left coronary artery for 30 minutes followed by reperfusion (N=3-6 per each group). IS was measured by triphenyl tetrazolium chloride (TTC) staining method at 1, 3 and 24 hours, and with serum cardiac troponin I (cTnI) levels 24 hours after surgery. Myocardial NLPR3 expression was quantified at 3, 6 and 24 hours of reperfusion. A novel pharmacologic NLRP3 inhibitor (NLRP3inh) or vehicle were administrated intraperitoneally at time of reperfusion, or with a delay of 1 or 3 hours. Results: IS measured at TTC was significantly larger at 24 hours (43±4% of the area at risk) vs 3 hours (30±5%) and 1 hour (11±2%, P&lt;0.001 for trend). NLRP3 myocardial expression was also significantly increased at 24 hours (1,097±382% of sham, P&lt;0.001) and 6 hours (928±380%, P&lt;0.001) vs 3 hours (111±15%). When compared with vehicle, the NLRP3inh given at reperfusion did not significantly reduce IS at 3 hours, while it significantly reduced IS at 24 hours (Figure). Administration of the NLRP3inh 1 hour after reperfusion significantly reduced IS at 24 hours, while it did not when given with a delay of 3 hours (Figure). Conclusions: Inhibition of the NLRP3 inflammasome within 1 hour of reperfusion limits the secondary inflammatory injury following myocardial ischemia-reperfusion.