BACKGROUND: Epiglottic masses are often asymptomatic, making them difficult to detect during preoperative examinations. Consequently, anesthesiologists may face ventilation difficulties with no apparent cause. Epiglottic masses can sometimes obstruct laryngoscope insertion into the epiglottic vallecula, complicating general anesthesia induction. In such cases, supraglottic airway insertion may be a viable alternative; however, the limited case reports on its use for epiglottic masses make its applicability unclear. Therefore, we test the hypothesis that a larger laryngeal artificial mass could obstruct the view of the larynx, even when supraglottic airways are used in a mannequin study. METHODS: We utilized an airway management simulator (Air Sim Multi®: Nihon 3B Scientific, Japan) to place various sizes of artificial masses (tumors) above the epiglottis. The groups included a control group with no mass, small size mass group, middle size mass group, and large size mass group. The supraglottic airway (i-gel®: Intersurgical, UK) was then inserted 10 times. We categorized the view of the vocal cords using a bronchoscope inserted through the tip of the cuff according to the Cormack-Lehane classification. In addition, we performed pressure-controlled ventilation, adjusting the inspiratory pressure from 10 cm H₂O to 25 cm H₂O, while measuring the tidal volumes. RESULTS: The Cormack-Lehane classification grade increased in correlation with the mass size. In each inspiratory pressure, tidal volume decreased in correlation with the mass size. Furthermore, in the large-size mass group, even at an inspiratory pressure of 25 cm H₂O, achieving the tidal volume required for general adult respiratory management was deemed difficult. CONCLUSION: In a mannequin study, we observed that epiglottic masses significantly increased the Cormack-Lehane classification grade and reduced tidal volume, with these effects correlating with the size of the mass. This finding suggests that the appropriateness of using a supraglottic airway may depend on the size and weight of the epiglottic mass.

Introduction The efficient clearance of bacteria by macrophages is crucial for the timely resolution of inflammation. In this study, we investigated the role of microRNA-21 (miR-21)-induced phagocytosis and its intracellular signaling pathways in human macrophages in vitro. Methods Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from 15 healthy volunteers. Differentiated human macrophages were incubated with lipopolysaccharide (LPS) to determine the time course of changes in phagocytic activity and miR-21 expression. The expression of candidate genes targeted by miR-21 and its downstream effectors was quantitatively assessed. The effects of miR-21 modulation were also examined via transfection with miR-21 mimics and inhibitors. Results Incubation of human macrophages with LPS upregulated both phagocytosis and miR-21 expression. Notably, changing miR-21 expression levels using miR-21 mimics or inhibitors led to significant and opposite changes in the expression of its downstream effectors. miR-21 induction in macrophages downregulated PDCD4 and PTEN, promoted the phosphorylation of Akt and the production of the anti-inflammatory cytokine IL-10, and facilitated phagocytosis. Conclusion This study directly confirms that LPS upregulates macrophage phagocytosis and miR-21 expression. Elevated miR-21 levels in macrophages enhanced phagocytosis, contributing to an anti-inflammatory phenotype. These findings underscore the importance of miR-21 in resolving inflammation.

BACKGROUND: Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism. OBJECTIVE: To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac and skeletal myopathy. METHODS AND RESULTS: High glucose promoted translocation of MondoA into the nucleus, which upregulated Arrdc4 transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of ARRDC4 was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial Arrdc4 expression in multiple types of mouse models of diabetes. We then analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. However, deletion of Arrdc4 augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that Arrdc4-knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type (WT) animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression (beyond levels observed during diabetes) using adenoassociated virus suggests that high levels of myocardial ARRDC4 have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the ARRDC4 mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1. CONCLUSIONS: ARRDC4 serves as a regulator of hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.

BACKGROUND: Arterial catheterisation in children can be challenging and time-consuming. We aimed to compare the success rates of ultrasound-guided arterial catheterisation utilising the short-axis out-of-plane approach with dynamic needle tip positioning in the radial, dorsalis pedis, and posterior tibial arteries in paediatric patients. We also examined the factors influencing the catheterisation success using dynamic needle tip positioning. METHODS: Paediatric patients (aged <3 yr) undergoing cardiac surgery were randomly assigned to three groups based on puncture sites: radial artery (Group R), dorsalis pedis artery (Group D), and posterior tibial artery (Group P). The first-attempt and overall success rates of arterial catheterisation were compared, followed by multiple logistic regression analysis (dependent variable: first-attempt success; independent variables: body weight, diameter and depth of the artery, targeted artery, and trisomy 21). RESULTS: The study included 270 subjects (n=90 per group). There was no significant difference in the first-attempt (Group R: 82%, Group D: 76%, and Group P: 81%) and overall success rates (Group R: 94%, Group D: 93%, and Group P: 91%) among the three groups. The diameter of the artery (per 0.1 mm) (odds ratio: 1.32, 95% confidence interval: 1.09-1.60) and trisomy 21 (odds ratio: 0.43, 95% confidence interval: 0.20-0.92) were independent predictors of first-attempt success or failure. CONCLUSION: The first-attempt and overall success rates of arterial catheterisation of the dorsalis pedis and posterior tibial arteries were not inferior to those in the radial artery when using dynamic needle tip positioning. These two lower extremity peripheral arteries present viable alternative catheterisation sites in paediatric patients. CLINICAL TRIAL REGISTRATION: UMIN000042847.

OBJECTIVES: To compare the efficacy of the ultrasound-guided approach with and without dynamic needle-tip positioning and the palpation technique regarding success for peripheral venous catheterization in children. DESIGN: A systematic review with network meta-analysis. SETTING: Databases of MEDLINE (via PubMed) and Cochrane Central Register of Controlled Trials. PARTICIPANTS: Patients (<18 years) undergoing peripheral venous catheter insertion. INTERVENTIONS: Randomized clinical trials were included to compare the following techniques: the ultrasound-guided short-axis out-of-plane approach with dynamic needle-tip positioning, the approach without dynamic needle-tip positioning, and the palpation technique. MEASUREMENTS AND MAIN RESULTS: The outcomes were first-attempt and overall success rates. Eight studies were included in the qualitative analyses. According to the estimate of network comparison, dynamic needle-tip positioning was associated with higher first-attempt (risk ratio [RR] 1.67; 95% CI 1.33-2.09) and overall success rates (RR 1.25; 95% CI 1.08-1.44) than palpation. The approach without dynamic needle-tip positioning was not associated with higher first-attempt (RR 1.17; 95% CI 0.91-1.49) and overall success rates (RR 1.10; 95% CI 0.90-1.33) than palpation. Compared to the approach without dynamic needle-tip positioning, dynamic needle-tip positioning was associated with a higher first-attempt success rate (RR 1.43; 95% CI 1.07-1.92), but not a higher overall success rate (RR 1.14; 95% CI 0.92-1.41). CONCLUSIONS: Dynamic needle-tip positioning is efficacious for peripheral venous catheterization in children. It would be better to include dynamic needle-tip positioning for the ultrasound-guided short-axis out-of-plane approach.

The efficacy of the short-axis out-of-plane (SA-OOP) approach with and without dynamic needle tip positioning (DNTP) remains unclear. This systematic review with network meta-analysis aimed to compare the success rate of arterial line insertion in children using the SA-OOP approach with and without DNTP and the palpation technique. We searched MEDLINE (via PubMed) and the Cochrane Central Register of Controlled Trials. We included randomized controlled trials that compared two of the following techniques for arterial line insertion in children: (1) the ultrasound-guided SA-OOP approach with DNTP; (2) the ultrasound-guided SA-OOP approach without DNTP; and (3) the palpation technique. A network meta-analysis was performed. The outcomes were first-attempt and overall success rates. Eight studies were finally included in this network meta-analysis. The ultrasound-guided SA-OOP approach with DNTP was associated with increased first-attempt (relative risk RR = 3.45 [95% confidence interval (CI) 2.51-4.74]) and overall success rates (RR = 1.81 [1.41-2.32]) when compared with palpation. The same approach performed without DNTP was also associated with increased first-attempt (RR = 1.96 [1.59-2.42]) and overall success rates (RR = 1.25 [1.05-1.49]) when compared with palpation. The ultrasound-guided SA-OOP approach with DNTP was associated with increased first-attempt (RR = 1.76 [1.26-2.44]) and overall success rates (RR = 1.45 [1.10-1.91]) when compared with the same approach performed without DNTP. DNTP should be performed during the ultrasound-guided SA-OOP approach for arterial line insertion in children, as this can help increase first attempt and overall success rates.

BACKGROUND: An ancient family of arrestin-fold proteins, termed alpha-arrestins, may have conserved roles in regulating nutrient transporter trafficking and cellular metabolism as adaptor proteins. One alpha-arrestin, TXNIP (thioredoxin-interacting protein), is known to regulate myocardial glucose uptake. However, the in vivo role of the related alpha-arrestin, ARRDC4 (arrestin domain-containing protein 4), is unknown. METHODS: We first tested whether interaction with GLUTs (glucose transporters) is a conserved function of the mammalian alpha-arrestins. To define the in vivo function of ARRDC4, we generated and characterized a novel Arrdc4 knockout (KO) mouse model. We then analyzed the molecular interaction between arrestin domains and the basal GLUT1. RESULTS: ARRDC4 binds to GLUT1, induces its endocytosis, and blocks cellular glucose uptake in cardiomyocytes. Despite the closely shared protein structure, ARRDC4 and its homologue TXNIP operate by distinct molecular pathways. Unlike TXNIP, ARRDC4 does not increase oxidative stress. Instead, ARRDC4 uniquely mediates cardiomyocyte death through its effects on glucose deprivation and endoplasmic reticulum stress. At baseline, Arrdc4-KO mice have mild fasting hypoglycemia. Arrdc4-KO hearts exhibit a robust increase in myocardial glucose uptake and glycogen storage. Accordingly, deletion of Arrdc4 improves energy homeostasis during ischemia and protects cardiomyocytes against myocardial infarction. Furthermore, structure-function analyses of the interaction of ARRDC4 with GLUT1 using both scanning mutagenesis and deep-learning Artificial Intelligence identify specific residues in the C-terminal arrestin-fold domain as the interaction interface that regulates GLUT1 function, revealing a new molecular target for potential therapeutic intervention against myocardial ischemia. CONCLUSIONS: These results uncover a new mechanism of ischemic injury in which ARRDC4 drives glucose deprivation-induced endoplasmic reticulum stress leading to cardiomyocyte death. Our findings establish ARRDC4 as a new scaffold protein for GLUT1 that regulates cardiac metabolism in response to ischemia and provide insight into the therapeutic strategy for ischemic heart disease.

Underlying molecular mechanisms for the development of diabetic cardiomyopathy remain to be determined. Long-term exposure to hyperglycemia causes oxidative stress, which leads to cardiomyocyte dysfunction. Previous studies established the importance of thioredoxin-interacting protein (Txnip) in cellular redox homeostasis and glucose metabolism. Txnip is a highly glucose-responsive molecule that interacts with the catalytic center of reduced thioredoxin and inhibits the antioxidant function of thioredoxin. Here, we show that the molecular interaction between Txnip and thioredoxin plays a pivotal role in the regulation of redox balance in the diabetic myocardium. High glucose increased Txnip expression, decreased thioredoxin activities, and caused oxidative stress in cells. The Txnip-thioredoxin complex was detected in cells with overexpressing wild-type Txnip but not Txnip cysteine 247 to serine (C247S) mutant that disrupts the intermolecular disulfide bridge. Then, diabetes was induced in cardiomyocyte-specific Txnip C247S knock-in mice and their littermate control animals by injections of streptozotocin (STZ). Prolonged hyperglycemia upregulated myocardial Txnip expression in both genotypes. The absence of Txnip's inhibition of thioredoxin in Txnip C247S mutant hearts promoted mitochondrial antioxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage by diabetes. Stress hemodynamic analysis uncovered that Txnip C247S knock-in hearts have a greater left ventricular contractile reserve than wild-type hearts under STZ-induced diabetic conditions. These results provide novel evidence that Txnip serves as a regulator of hyperglycemia-induced cardiomyocyte toxicities through direct inhibition of thioredoxin and identify the single cysteine residue in Txnip as a therapeutic target for diabetic injuries.NEW & NORTEWORTHY Thioredoxin-interacting protein (Txnip) has been of great interest as a molecular mechanism to mediate diabetic organ damage. Here, we provide novel evidence that a single mutation of Txnip confers a defense mechanism against myocardial oxidative stress in streptozotocin-induced diabetic mice. The results demonstrate the importance of Txnip as a cysteine-containing redox protein that regulates antioxidant thioredoxin via disulfide bond-switching mechanism and identify the cysteine in Txnip as a therapeutic target for diabetic cardiomyopathy.

RATIONALE: Thioredoxin-interacting protein (Txnip) is a novel molecular target with translational potential in diverse human diseases. Txnip has several established cellular actions including binding to thioredoxin, a scavenger of reactive oxygen species (ROS). It has been long recognized from in vitro evidence that Txnip forms a disulfide bridge through cysteine 247 (C247) with reduced thioredoxin to inhibit the anti-oxidative properties of thioredoxin. However, the physiological significance of the Txnip-thioredoxin interaction remains largely undefined in vivo. OBJECTIVE: A single mutation of Txnip, C247S, abolishes the binding of Txnip with thioredoxin. Using a conditional and inducible approach with a mouse model of a mutant Txnip that does not bind thioredoxin, we tested whether the interaction of thioredoxin with Txnip is required for Txnip's pro-oxidative or cytotoxic effects in the heart. METHODS AND RESULTS: Overexpression of Txnip C247S in cells resulted in a reduction in ROS, due to an inability to inhibit thioredoxin. Hypoxia (1% O2, 24 h)-induced killing effects of Txnip were decreased by lower levels of cellular ROS in Txnip C247S-expressing cells compared with wild-type Txnip-expressing cells. Then, myocardial ischemic injuries were assessed in the animal model. Cardiomyocyte-specific Txnip C247S knock-in mice had better survival with smaller infarct size following myocardial infarction (MI) compared to control animals. The absence of Txnip's inhibition of thioredoxin promoted mitochondrial anti-oxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage induced by MI. Furthermore, an unbiased RNA sequencing screen identified that hypoxia-inducible factor 1 signaling pathway was involved in Txnip C247S-mediated cardioprotective mechanisms. CONCLUSION: Txnip is a cysteine-containing redox protein that robustly regulates the thioredoxin system via a disulfide bond-switching mechanism in adult cardiomyocytes. Our results provide the direct in vivo evidence that regulation of redox state by Txnip is a crucial component for myocardial homeostasis under ischemic stress.

Peripheral vascular catheterization (PVC) in pediatric patients is technically challenging. Ultrasound guidance has gained the most interest in perioperative and intensive care fields because it visualizes the exact location of small target vessels and is less invasive than other techniques. There have been a growing number of studies related to ultrasound guidance for PVC with or without difficult access in pediatric patients, and most findings have demonstrated its superiority to other techniques. There are various ultrasound guidance approaches, and a comprehensive understanding of the basics, operator experience, and selection of appropriate techniques is required for the successful utilization of this technique. This narrative review summarizes the literature regarding ultrasound-guided PVC principles, approaches, and pitfalls to improve its clinical performance in pediatric settings.

OBJECTIVES: This study evaluated whether the dynamic needle tip positioning technique increased the success rate of ultrasound-guided peripheral venous catheterization in pediatric patients with a small-diameter vein compared with the static ultrasound-guided technique. DESIGN: Randomized controlled study. SETTING: Single institution, Osaka Women's and Children's Hospital. PATIENTS: The study population included 60 pediatric patients less than 2 years old who required peripheral venous catheterization in the PICU. INTERVENTIONS: Patients were randomly divided into the dynamic needle tip positioning (n = 30) or static group (n = 30). Each group received ultrasound-guided peripheral venous catheterization with or without dynamic needle tip positioning, respectively. The Fisher exact test, Kaplan-Meier curve plots, log-rank tests, and Mann-Whitney U test were used in the statistical analysis. MEASUREMENTS AND MAIN RESULTS: The first-attempt success rate was higher in the dynamic needle tip positioning group than in the static group (86.7% vs 60%; p = 0.039; relative risk = 1.44; 95% CI, 1.05-2.0). The overall success rate within 10 minutes was higher in the dynamic needle tip positioning group than in the static group (90% vs 63.3%; p = 0.03; relative risk = 1.42; 95% CI, 1.06-1.91). Significantly fewer attempts were made in the dynamic needle tip positioning group than in the static group (median [interquartile range, range] = 1 [1-1, 1-2] vs 1 [1-2, 1-3]; p = 0.013]). The median (interquartile range) catheterization times were 51.5 seconds (43-63 s) and 71.5 seconds (45-600 s) in the dynamic needle tip positioning and static groups, respectively (p = 0.01). CONCLUSIONS: Dynamic needle tip positioning increased the first-attempt and overall success rates of ultrasound-guided peripheral venous catheterization in pediatric patients less than 2 years old.

BACKGROUND: Recombinant activated factor VII (rFVIIa) concentrate reduces allogeneic blood transfusions, but it may increase thromboembolic complications in complex cardiac surgery. The mixture of activated factor VII (FVIIa) and factor X (FX) (FVIIa/FX) (FVIIa:FX = 1:10) is a novel bypassing agent for hemophilia patients. We hypothesized that the combination of FX and FVIIa could improve thrombin generation (TG) in acquired multifactorial coagulation defects such as seen in cardiac surgery and conducted in vitro evaluation of FVIIa/FX in parallel with other coagulation factor concentrates using in vitro and in vivo diluted plasma samples. METHODS: Plasma samples were collected from 9 healthy volunteers and 12 cardiac surgical patients. We measured TG (Thrombinoscope) using in vitro 50% dilution plasma and in vivo dilution plasma after cardiopulmonary bypass, in parallel with thromboelastometry (ROTEM) and standard coagulation assays. In vitro additions of FVIIa/FX (0.35, 0.7, and 1.4 μg/mL, based on the FVIIa level), rFVIIa (1.4, 2.8, and 6.4 μg/mL), prothrombin complex concentrate (0.3 international unit), and 20% plasma replacement were evaluated. RESULTS: In diluted plasma, the addition of either FVIIa/FX or rFVIIa shortened the lag time and increased the peak TG, but the effect in lag time of FVIIa/FX at 0.35 μg/mL was more extensive than rFVIIa at 6.4 μg/mL. Prothrombin complex concentrate increased peak TG by increasing the prothrombin level but failed to shorten the lag time. No improvement in any of the TG variables was observed after 20% volume replacement with plasma. The addition of factor concentrates normalized prothrombin time/international normalized ratio but not with plasma replacement. In cardiac patients, similar patterns were observed on TG in post-cardiopulmonary bypass samples. FVIIa/FX shortened clotting time (CT) in a concentration-dependent manner on CT on thromboelastometry. Plasma replacement did not improve CT, but a combination of plasma and FVIIa/FX (0.35 μg/mL) more effectively shortened CT than FVIIa/FX alone. CONCLUSIONS: The combination of FVIIa and FX improved TG more efficiently than rFVIIa alone or plasma in dilutional coagulopathy models. The required FVIIa dose in FVIIa/FX was considerably lower than those reported during bypassing therapy in hemophilia patients (1.4-2.8 μg/mL). The combination of plasma could restore coagulation more efficiently compared to FVIIa/FX alone. Lesser FVIIa requirement to exert procoagulant activity may be favorable in terms of reducing systemic thromboembolic complications.

BACKGROUND: Macrophage phagocytosis constitutes an essential part of the host defence against microbes and the resolution of inflammation. Hyperglycaemia during sepsis is reported to reduce macrophage function, and thus, potentiate inflammatory deterioration. We investigated whether high-glucose concentrations augment lipopolysaccharide-induced reduction in macrophage phagocytosis via the endoplasmic stress-C/EBP homologous protein (CHOP) pathway using animal and laboratory investigations. METHODS: Peritoneal macrophages of artificially ventilated male Wistar rats, divided into four groups based on target blood glucose concentrations achieved by glucose administration with or without lipopolysaccharide, were obtained after 24 h. Human macrophages were also cultured in normal or high glucose with or without lipopolysaccharide exposure for 72 h. Changes in the phagocytic activity, intranuclear CHOP expression, and intracellular Akt phosphorylation status of macrophages were evaluated. These changes were also evaluated in human macrophages after genetic knock-down of CHOP by specific siRNA transfection or resolvin D2 treatment. RESULTS: Lipopolysaccharide impaired phagocytosis, increased intranuclear expression of CHOP, and inhibited Akt phosphorylation in both rat peritoneal and human macrophages. Hyperglycaemic glucose concentrations augmented these changes. Genetic knock-down of CHOP restored phagocytic ability and Akt phosphorylation in human macrophages. Furthermore, resolvin D2 co-incubation restored the inhibited phagocytosis and Akt phosphorylation along with the inhibition of intranuclear CHOP expression in human macrophages. CONCLUSIONS: These findings imply that controlling endoplasmic reticulum stress might provide new strategies for restoring reduced macrophage phagocytosis in sepsis-induced hyperglycaemia.

OBJECTIVE: Arterial catheterization for infants and small children is technically challenging. This study evaluated whether the dynamic needle tip positioning (DNTP) technique improved the success rate of ultrasound-guided radial artery catheterization in patients with a radial artery depth ≥4 mm compared with the conventional ultrasound-guided technique. DESIGN: Randomized controlled study. SETTING: Single institution, Osaka Women's and Children's Hospital. PARTICIPANTS: Patients (n = 40; age <3 years) with artery depth ≥4 mm. INTERVENTIONS: Patients were divided randomly into 2 groups. The DNTP group received ultrasound-guided radial artery catheterization with DNTP; the conventional group received catheterization without DNTP. MEASUREMENTS AND MAIN RESULTS: First-attempt success rates were 85% and 50% in the DNTP and conventional groups, respectively (p = 0.018; relative risk = 1.7; 95% CI: 1.06-2.73). Overall success rates within 10 minutes were 95% and 60% in the DNTP and conventional groups, respectively (p = 0.008; relative risk = 1.58; 95% CI: 1.09-2.3). Posterior wall puncture rates were 5% and 50% in the DNTP and conventional groups, respectively (p = 0.0014; relative risk = 0.1; 95% CI: 0.014-0.71). Significantly fewer attempts were made in the DNTP group (median = 1 v 1.5; p = 0.01). The median catheterization times were 38 seconds (34-55.5) and 149 seconds (49.5-600) in the DNTP and conventional groups, respectively (p = 0.0003). CONCLUSION: Dynamic needle tip positioning improved first-attempt and overall success rates of ultrasound-guided radial artery catheterization in pediatric patients with a radial artery depth ≥4 mm.

MicroRNAs (miRNAs) are small RNA molecules that modulate gene and protein expression in hematopoiesis. Platelets are known to contain a fully functional miRNA machinery. While platelets used for transfusion are normally stored at room temperature, recent evidence suggests more favorable effects under a cold-storage condition, including higher adhesion and aggregation properties. Thus, we sought to determine whether functional differences in platelets are associated with the differential profiling of platelet miRNA expressions. To obtain the miRNA expression profile, next-generation sequencing was performed on human platelets obtained from 10 healthy subjects. The miRNAs were quantified after being stored in three different conditions: 1) baseline (before storage), 2) stored at 22°C with agitation for 72 h, and 3) stored at 4°C for 72 h. Following the identification of miRNAs by sequencing, the results were validated at the level of mature miRNAs from 18 healthy subjects, by using quantitative polymerase chain reaction (qPCR). Differential expression was observed for 125 miRNAs that were stored at 4°C and 9 miRNAs stored at 22°C as compared to the baseline. The validation study by qPCR confirmed that storage at 4°C increased the expression levels (fold change 95% CI) of mir-20a-5p (1.87, p<0.0001), mir-10a-3p (1.88, p<0.0001), mir-16-2-3p (1.54, p<0.01), and mir-223-5p (1.38, p<0.05), compared with those of the samples stored at 22°C. These results show that miRNAs correlate with platelet quality under specific storage conditions. The data indicate that miRNAs could be potentially used as biomarkers of platelet quality.

OBJECTIVE: Point-of-care (POC) devices allow for prothrombin time/international normalized ratio (PT/INR) testing in whole blood (WB) and timely administration of plasma or prothrombin complex concentrate during cardiopulmonary bypass surgery. This study evaluated the sensitivities of a new POC PT test, a dry-hematology method with heparin neutralization technology (DRIHEMATO PT-S [DRI PT-S]; A&T Corporation, Kanagawa, Japan), and compared it with other POC tests currently available. DESIGN: Prospective, observational study. SETTING: University hospital, single center. PARTICIPANTS: Healthy volunteers and warfarin-treated and cardiac surgical patients. MEASUREMENT AND MAIN RESULTS: In WB samples obtained from 6 healthy volunteers, PT-INR results of DRI PT-S were not affected by an in vitro addition of heparin <6.0 U/mL. In warfarin-treated samples (n = 88, PT/INR 0.98-3.87), PT-INR with DRI PT-S showed acceptable correlation with the laboratory method (r2 = 0.85, p < 0.001). In blood samples obtained from cardiac surgical patients (n = 72), heparin prolonged the PT/INR with the laboratory assay, dry-hematology method with non heparin neutralization technology (DRI PT), Coaguchek XS (Roche Diagnostics, Basel, Switzerland), and Hemochron Jr. (Accriva Diagnostics, Edison, NJ), but DRI PT-S was not affected by heparin anticoagulation. In nonheparinized samples, different methods between DRI PT-S and the laboratory method yielded acceptable correlations (r2 = 0.76, p < 0.0001). There was a moderate correlation between factor levels and the PT-INR with DRI PT-S (factor [F]II: r2 = 0.63, FVII: r2 = 0.47, FX: r2 = 0.67; p < 0.0001). CONCLUSIONS: This study demonstrated that PT/INR can be accurately assessed using the dry-hematology method in WB under therapeutic heparin levels. Currently available other POC PT/INR tests are affected by heparin, and thus they are not recommended for coagulation monitoring during cardiopulmonary bypass.

OBJECTIVES: Platelet defect mechanisms after cardiopulmonary bypass remain unclear. Our hypothesis microRNA expressions in circulating platelets significantly change between pre and post cardiopulmonary bypass, and consequent messenger RNA and protein expression level alterations cause postcardiopulmonary bypass platelet defect. DESIGN: Single-center prospective observational study. SETTING: Operating room of Kyoto Prefectural University of Medicine. PATIENTS: Twenty-five adult patients scheduled for elective cardiac surgeries under cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In the initial phase, changes in microRNA expression between pre and post cardiopulmonary bypass underwent next generation sequencing analysis (10 patients). Based on the results, we focused on changes in mir-10b and mir-96, which regulate glycoprotein 1b and vesicle-associated membrane protein 8, respectively, and followed them until messenger RNA and protein syntheses (15 patients) using quantitative polymerase chain reaction and Western blotting. Seven microRNAs including mir-10b and mir-96 exhibited significant differences in the initial phase. In the subsequent phase, mir-10b-5p and mir-96-5p overexpressions were confirmed, and glycoprotein 1b and vesicle-associated membrane protein 8 messenger RNA levels were significantly decreased after cardiopulmonary bypass: fold differences (95% CI): mir-10b-5p: 1.35 (1.05-2.85), p value equals to 0.01; mir-96-5p: 1.59 (1.06-2.13), p value equals to 0.03; glycoprotein 1b messenger RNA: 0.46 (0.32-0.60), p value of less than 0.001; and vesicle-associated membrane protein messenger RNA: 0.70 (0.56-0.84), p value of less than 0.001. Glycoprotein 1b and vesicle-associated membrane protein 8 were also significantly decreased after cardiopulmonary bypass: glycoprotein 1b: 82.6% (71.3-93.8%), p value equals to 0.005; vesicle-associated membrane protein 8: 79.0% (70.7-82.3%), p value of less than 0.001. CONCLUSIONS: Expressions of several microRNAs in circulating platelets significantly changed between pre and post cardiopulmonary bypass. Overexpressions of mir-10b and mir-96 decreased glycoprotein 1b and vesicle-associated membrane protein 8 messenger RNA as well as protein, possibly causing platelet defect after cardiopulmonary bypass.

BackgroundChildren with cyanotic heart disease develop secondary erythrocytosis and thrombocytopenia via unknown mechanisms. Mature erythrocyte microRNAs may reflect clinical pathologies and cell differentiation processes pre-enucleation. This study evaluated erythrocyte microRNAs in children with cyanotic heart disease.MethodsErythrocyte microRNAs from children with cyanotic and acyanotic heart disease and without cardiac disease were quantified with Ion PGM System (n=10 per group). Differential expression was confirmed by quantitative PCR (qPCR; n=20 per group).ResultsMir-486-3p, mir-486-5p, and mir-155-5p increased in patients with cyanotic heart disease compared with those without heart disease: fold differences (95% confidence interval): mir-486-3p: 1.92 (1.14-3.23), P=0.011; mir-486-5p: 2.27 (1.41-3.65), P<0.001; and mir-155-5p: 1.44 (1.03-2.03), P=0.028. Mir-486-5p was increased, and let-7e-5p and mir-1260a were decreased in patients with acyanotic heart disease compared with those without heart disease: mir-486-5p: 1.66 (1.03-2.66), P=0.035; let-7e-5p: 0.66 (0.44-0.99), P=0.049; and mir-1260a: 0.53 (0.29-0.99), P=0.045.ConclusionSeveral microRNA levels changed in children with cyanotic and acyanotic heart disease. Mir-486-3p and -5p are associated with hematopoietic differentiation. Mir-486-3p regulates the erythroid vs. megakaryocyte lineage fate decision. Mir-155 is a hypoxia-inducible microRNA, whose overexpression inhibits megakaryocyte differentiation. Erythrocyte microRNA expression changes may contribute to erythrocytosis and thrombocytopenia in children with cyanotic heart disease.

Background: Anucleate platelets can undergo apoptosis in response to various stimuli, as do nucleated cells. Cardiopulmonary bypass (CPB) causes platelet dysfunction and can also activate platelet apoptotic pathways. We therefore evaluated time-dependent changes in blood platelet Bax (a pro-apoptotic molecule) levels and platelet dysfunction after cardiac surgery. Methods: We assessed blood samples obtained from subjects having on-pump or off-pump coronary artery bypass graft surgery ( n =20 each). We also evaluated the in vitro effects of platelet Bax increase in eight healthy volunteers. Results: Thrombin-induced platelet calcium mobilisation and platelet-surface glycoprotein Ib (GPIb) expression were lowest at weaning from CPB and did not recover on postoperative day one. On-pump surgery increased platelet expression of Bax, especially the oligomerised form, along with translocation of Bax from the cytosol to mitochondria and platelet-surface tumour necrosis factor-alpha (TNF-α)-converting enzyme (TACE) expression. In contrast, mitochondrial cytochrome c expression was reduced. While similar in direction, the magnitude of the observed changes was smaller in patients having off-pump surgery. In vitro , a cell-permeable Bax peptide increased platelet Bax expression to the same extent seen during bypass and produced similar platelet changes. These apoptotic-like changes were largely reversed by Bcl-xL pre-administration, and were completely reversed by combined application of inhibitors that stabilise outer mitochondrial membrane permeability and TACE. Conclusions: CPB increases platelet Bax expression, which contributes to reduced platelet-surface GPIb expression and thrombin-induced platelet calcium changes. These changes in platelet apoptotic signalling might contribute to platelet dysfunction after CPB. Clinical trial registration: UMIN Clinical Trials Registry (number UMIN000006033).

BACKGROUND: The main cause of unsuccessful peripheral radial artery catheterization using traditional palpation is imprecisely locating the arterial center. The authors evaluated factors causing disparities between the arterial centers determined by palpation versus ultrasound. The authors applied them to create and test a novel catheterization training program. METHODS: The arterial central axis was determined by ultrasound and palpation in 350 adults. Potential independent predictors of disparity included sex, body mass index, pulse pressure, transverse arterial diameter, subcutaneous arterial depth, chronic hypertension, and experience as an anesthesiologist (less than 3 vs. greater than or equal to 3 yr). Using the results, the authors developed a radial artery catheterization training program. It was tested by enrolling 20 first-year interns, randomized to a training or control group. The time to successful insertion was the primary outcome measure. The success rate and time required for catheterization by palpation were evaluated in 100 adult patients per group. RESULTS: Independent predictors of central axis disparity were pulse pressure, subcutaneous radial artery depth, years of experience, and chronic hypertension. Training improved the catheterization time (training group 56 ± 2 s vs. control group 109 ± 2 s; difference -53 ± 3 s; 95% CI, -70 to -36 s; P < 0.0001) and total success rate (training group 83 of 100 attempts, 83%; 95% CI, 75 to 90 vs. control group 57 of 100, 57%; 95% CI, 47 to 66; odds ratio, 3.7; 95% CI, 2.7 to 5.1). CONCLUSIONS: Misjudging the central axis position of the radial artery is common with a weak pulse and/or deep artery. The authors' program, which focused on both these issues, shortened the time for palpation-guided catheterization and improved success.

INTRODUCTION: Venous catheterisation in paediatric patients can be technically challenging. We examined factors affecting catheterisation of invisible and impalpable peripheral veins in children and evaluated the best site for ultrasound-guided catheterisation. METHODS: Systolic pressure, age, sex, and American Society of Anaesthesiologists (ASA) physical status were determined in 96 children weighing less than 20 kg. Vein diameter and subcutaneous depth were measured with ultrasound. Logistic regression was used to evaluate the contribution of these factors to cannulation success with (n = 65) or without (n = 31) ultrasound guidance. Thereafter, we randomly assigned 196 patients for venous catheter insertion in the dorsal veins of the hand, the cephalic vein in the forearm, or the great saphenous vein. Success rates and vein diameters were evaluated by using Dunn tests; insertion time was evaluated by using Kaplan-Meier cumulative incidence analysis. RESULTS: Independent predictors of catheterisation were ultrasound guidance (odds ratio (OR) = 7.3, 95% confidence interval (CI) 2.0 to 26.0, P = 0.002), vein diameter (OR = 1.5 per 0.1 mm increase in diameter, 95% CI 1.1 to 2.0, P = 0.007), and ASA physical status (OR = 0.4 per status 1 increase, 95% CI 0.2 to 0.9, P = 0.03). Cephalic veins were significantly larger (cephalic diameter 1.8 mm, P = 0.001 versus saphenous 1.5 mm, P <0.001 versus dorsal 1.5 mm). Catheterisation success rates were significantly better at the cephalic vein than either the dorsal hand or saphenous vein (cephalic 95%, 95% CI 89% to 100%, P <0.001 versus dorsal 69%, 95% CI 56% to 82%, P = 0.03 versus saphenous 75%, 95% CI 64% to 86%). CONCLUSIONS: The cephalic vein in the proximal forearm appears to be the most appropriate initial site for ultrasound-guided catheterisation in invisible and impalpable veins of paediatric patients. TRIAL REGISTRY NUMBER: UMIN Clinical Trials Registry as UMIN000010961. Registered on 14 June 2013.

BACKGROUND: Thromboelastometric evaluation of coagulation might be useful for prediction and management of bleeding after paediatric cardiac surgery. We tested the hypothesis that the use of a thromboelastometry-guided algorithm for blood product management reduces blood loss and transfusion requirements. METHODS: We studied 78 patients undergoing paediatric cardiac surgery with cardiopulmonary bypass (CPB) for the initial 12 h after operation. Stepwise multiple linear regression was used to develop an algorithm to guide blood product transfusions. Thereafter, we randomly assigned 100 patients to conventional or algorithm-guided blood product management, and assessed bleeding and red cell transfusion requirements. RESULTS: CPB time, post-bypass rotational thromboelastometry (ROTEM(®)) EXTEM amplitude at 10 min (A10), and FIBTEM-A10 were independently associated with chest tube drainage volume during the initial 12 h after operation. Discriminative analysis determined cut-off values of 30 mm for EXTEM-A10 and 5 mm for FIBTEM-A10, and estimated optimal intraoperative fresh-frozen plasma and platelet concentrate transfusion volumes. Thromboelastometry-guided post-bypass blood product management significantly reduced postoperative bleeding (9 vs 16 ml kg(-1), P<0.001) and packed red cell transfusion requirement (11 vs 23 ml kg(-1), P=0.005) at 12 h after surgery, and duration of critical care stay (60 vs 71 h, P=0.014). CONCLUSIONS: Rotational thromboelastometry-guided early haemostatic intervention by rapid intraoperative correction of EXTEM-A10 and FIBTEM-A10 reduced blood loss and red cell transfusion requirements after CPB, and reduced critical care duration in paediatric cardiac surgical patients. CLINICAL TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000006832 (December 4, 2011).

BACKGROUND: An accurate and rapid determination of fibrinogen level is important during hemorrhage to establish a timely hemostatic intervention. The accuracy of fibrinogen measurements may be affected by the specific methodology for its determination, fluid therapies, and anticoagulant agents. The dry-hematology method (DRIHEMATO®) is a novel approach to determine fibrinogen levels in plasma and whole blood based on thrombin-activated coagulation time. We hypothesized that plasma or whole blood fibrinogen level using the dry-hematology method would be similar to those measured with conventional plasma fibrinogen assays. METHODS: Acquired hypofibrinogenemia was modeled by serial dilutions of blood samples obtained from 12 healthy volunteers. Citrated whole blood samples were diluted with either normal saline, 5% human albumin, or 6% hydroxyethyl starch to achieve 25%, 50%, and 75% volume replacement. The dry-hematology method, the Clauss method, the prothrombin time (PT)-derived method, determination of antigen levels, and thromboelastometric fibrin formation were compared in plasma or whole blood samples. The effect of heparin on each assay was examined (0 to 6 IU/mL). Comparisons of dry-hematology and other methods were also conducted using ex vivo samples obtained from cardiac surgical patients (n = 60). RESULTS: In plasma samples, there were no significant differences between dry-hematology and the Clauss method, while dry-hematology showed lower fibrinogen levels compared with PT-derived and antigen level methods. The dry-hematology method yielded acceptable concordance correlation coefficients (Pc) with the Clauss method, the PT-derived method, and fibrinogen antigen levels (Pc = 0.91-0.99). The type of diluents and heparin affected the results of the PT-derived method and thromboelastometric assay, but not the dry-hematology method. In cardiac surgical patients, the overall correlation in fibrinogen levels between dry-hematology and the other methods was comparable to the results from in vitro dilution experiments. The dry-hematology reported higher fibrinogen values in whole blood compared with those measured in plasma samples, but hematocrit adjustment decreased the bias between whole blood and plasma samples from 73 mg/dL (95% prediction interval: 40, 106) to -13 mg/dL (95% prediction interval: -35, 8.5). CONCLUSIONS: This study demonstrated that fibrinogen levels can be accurately assessed by the dry-hematology method in plasma and the results are not affected by heparin or colloids. For whole blood fibrinogen measurements by dry-hematology, hematocrit adjustment is necessary to compensate for dynamic changes in hematocrit in perioperative bleeding settings.

BACKGROUND: Radial arterial catheterization in pediatric patients is occasionally difficult despite ultrasound guidance. We therefore assessed the factors affecting catheterization and tested an intervention designed to improve its success. METHODS: For initial assessment, we performed multiple logistic regression analyses using 102 pediatric patients. Dependent variables included first-attempt and overall success or failure; independent variables were systolic blood pressure, weight, ASA physical status, trisomy 21, arterial diameter, and subcutaneous depth of the radial artery (<2, 2-4, ≥4 mm). The effect of subcutaneous arterial depth on cannulation success was assessed using Kaplan-Meier curves with log-rank and Dunn tests. We then assessed catheterization success in 60 patients who were randomized to no treatment or subcutaneous saline injection, as necessary, to increase the subcutaneous arterial depth from <2 to 2 to 4 mm. RESULTS: Subcutaneous arterial depth of 2 to 4 mm was derived as a significant independent predictor of initial and overall success from the multiple logistic regression analyses. The 2 to 4 mm group had a significantly shorter catheterization time compared with the other 2 groups in the log-rank test (2-4 vs <2 mm group; P = 0.01, 2-4 vs ≥4 mm group; P < 0.001), and higher success rate in the first attempt (<2 [43.8%] vs 2-4 mm [76.9%], P = 0.02; 2-4 [76.9%] vs ≥4.0 mm [19.4%], P < 0.001), and the overall attempt (<2 [62.5%] vs 2-4 mm [89.7%], P = 0.04; 2-4 [89.7%] vs ≥4.0 mm [51.6%], P = 0.002). Injecting subcutaneous saline to bring arterial depth from <2 mm to 2 to 4 mm significantly shortened catheterization time (P = 0.002), and improved the success rate in the first-attempt (saline injection [85.0%] vs <2 mm [30.0%], P < 0.001), and the overall attempt (saline injection [90.0%] vs <2 mm [55.0%], P = 0.02). CONCLUSIONS: Ultrasound-guided radial artery catheterization in pediatric patients was fastest and most reliable when the artery was 2 to 4 mm below the skin surface. For arteries located <2 mm below the skin surface, increasing the depth to 2 to 4 mm by subcutaneous saline injection reduced catheterization time and improved the success rate.

PURPOSE: The Risk Adjustment for Congenital Heart Surgery (RACHS-1) classification was originally designed to facilitate the prediction of in-hospital mortality for pediatric cardiac surgery patients. However, there have been few reports on clinical outcomes predicted by the RACHS-1 category, especially in an Asian population. The aim of this study was to determine whether RACHS-1 classification can predict patient outcomes. METHODS: A total of 580 pediatric cardiac surgery procedures performed from January 2005 to December 2009 were retrospectively classified into the six RACHS-1 categories. The association between RACHS-1 category and clinical outcomes, including length of catecholamine requirement, mechanical ventilation time, intensive care unit stay, and in-hospital mortality, were examined. RESULTS: The frequencies of RACHS-1 categories in the study population were: category 1, 10.7 %; category 2, 36.7 %; category 3, 42.8 %; category 4, 6.6 %; category 5, 0.0 %; category 6, 3.3 %. There was a significant linear correlation between RACHS-1 category and in-hospital mortality (r = 0.96, p < 0.001). Kaplan-Meier analysis demonstrated that length of catecholamine infusion, mechanical ventilation time, and ICU stay were significantly different (p < 0.05) in the different RACHS-1 categories, except for those between category 4 and 6 (p = 0.09). CONCLUSIONS: Based on the results of our analysis, we conclude that the RACHS-1 stratification system can predict in-hospital mortality and patient outcomes in patients undergoing pediatric cardiac surgery.

過去の研究報告で、妊娠高血圧症候群患者の胎盤及び血液中で変化したmiRNAのmimicと、そのmiRNAに特異的なmiRNA阻害薬を、ヒト血管内皮細胞（HUVEC, HMEC-1）に遺伝子導入し、miRNAのターゲットとなる（相補配列を有する）mRNAとタンパク質の発現が変化することを、培養細胞実験で確認した。 臨床研究では、コロナ禍のために正常妊娠および妊娠高血圧症妊婦被験者のリクルートに難を要する状況が継続し、可能な範囲で研究を継続してきた。次世代シーケンサーを施行するための人数をようやく得ることができ、外部委託業者にその網羅的解析を依頼した。現在、その研究結果待ちの状態である。

耐糖能異常を有する病態では、白血球機能低下から感染防御能の低下に発展することが知られている。近年、小胞体ストレス とグルコースホメオスタシスの関連が示唆されるようになったことから、我々は、白血球の中でも感染制御に重要な役割を占めるマクロファージに着眼した。研究の結果、敗血症高血糖ストレス状況下では、小胞体ストレスの増強がCHOP経路を介し、マクロファージの機能障害の一因となることが明らかとなった。

昨年度、次世代シーケンサーによる網羅的解析およびvalidation PCRの結果から、Mir-486-3p、 mir-486-5p、mir-155-5p がチアノーゼ群で有意に上昇していることが明らかとなった。 また、非チアノーゼ群では、 Mir-486-5pが有意に上昇し、let-7e-5p および mir-1260a が有意に低下していることが明らかとなった。miR-486 (-3p、-5p)、miR-155は血球分化に深く関わることが報告されている。特に、miR-486-3pの上昇は巨核球・赤芽球前駆細胞に作用し、赤芽球系への分化を促進させ、巨核球系への分化を抑制する。また、miR-155の上昇は巨核球系前駆細胞の分化を抑制する。したがって、これらのmiRNAがチアノーゼ先天性心疾患患者の血球異常症の発症メカニズムに強く関与していることが示唆された。 本年度は、今後は得られたデータをさらに詳細な臨床データと結びつけること、また、vitro実験系において、低酸素刺激において赤芽球系の細胞でこれらのmiRNA変化が誘導されること、また、動物モデルの構築（左ー右シャント、右ー左シャント あるいは、hypoxic chamberによる低酸素誘導）を模索することを主眼に研究を計画していたが、研究代表者の海外留学に伴う資格喪失のため中断せざるを得なくなった。

本研究では、AKIを発症しやすい心臓血管手術患者周術期や、集中治療を要する重症症例の尿中、血液中からエクソソーム、クロマチンを抽出し、次世代シーケンサーを用いてmiRNA、及びceRNA発現の網羅的解析を行った。具体的には、RIFLE 及びAKIN criteriaを用いて、周術期のAKIの重症度をステージ分類し、急性腎障害時に変化するmiRNAおよびceRNAの発現を多変量解析により選出した。現在、統計的有意差を得るため、症例収集している。さらに、データベースを用いてmiRNA-ceRNA, miRNA-ncRNAのネットワークを探索する事に、主眼を置いた。 今回使用する次世代シーケンサーは、網羅的なmiRNAプロファイリングを未知のnon-cording RNAの発現に対してだけでなく、miRNA発現の絶対的定量評価が行える点で、従来のマイクロアレイ法と比べ、感度、正確性、再現性などにおいて優れている。近年、miRNAは抗体医薬と異なり開発に特別なノウハウが不必要なため、遺伝子治療として核酸創薬の対象になっている。早期診断マーカーに有用性の高いmiRNAのターゲットが、急性腎障害抑制に有用なタンパク質であった場合、今後の遺伝子治療に展開出来る可能性があると考えている。 また、一方で研究手法が次世代シーケンサーを用いた研究手法で同様であるため、共同研究を行った研究課題”次世代シーケンサーを用いた、チアノーゼ先天性心疾 患における血球異常症の発症メカニズムに関与する赤血球中microRNAの網羅的解析”に関してh、共同演者として2017年度の日本麻酔科学会で報告し、最優秀演題を受賞した。さらに2018年度の日本麻酔科学会においても、”保存赤血球製剤中における赤血球内microRNA経時変化の網羅的解析”を報告した。

心臓血管手術患者を対象に、周術期の急性腎障害（AKI: Acute Kidney Injury）発症の早期診断バイオマーカーの探索を目的に、血液中、尿中エクソソーム中のmicroRNA(miRNA)を網羅的に解析し、AKI発症の早期検出に特異度、感度何れも優れているmiRNAを検討した。また、統計解析で有意に変化があると判明したmiRNAのターゲットとなるタンパク質を同定する事で、AKI発症の新しい機序、及びmiRNAのAKI発症における役割を探求することを主眼において研究を行った。

人工心肺下心臓手術後の血小板機能低下の機序は、未だ解明の余地がある。無核細胞の血小板にも、種々の刺激で、有核細胞同様にアポトーシスが誘導される。人工心肺下手術周術期に起きる血小板機能障害の原因として、アポトーシスに関連する細胞内情報伝達系が関与している可能性があるとの仮説で実験を行った。血小板は、また、転写、翻訳機能を有し、RNAを含有している。本研究において、幾つかのmicroRNAが周術期に変化し、その結果、ターゲットとなるmRNAとタンパク質が低下することで、血小板の機能低下をもたらすとの仮説の元、実験を行った。手法として、次世代シーケンサーでmicroRNAの変化を網羅的に解析した。

現在、患者予後と明らかな関連があると報告が認められる血小板機能モニタリングは、ごく僅かである。 今回、我々は、周術期抗血小板薬効モニタリングのために有用な血小板由来microRNA明らかにするために研究を行った。まず、第一段階として、薬物及びずり応力を用いた血小板刺激を行い、最も鋭敏に反応するmicroRNAを同定することを行った。実際には、刺激前後の血小板由来microRNAを次世代シーケンサーを用いて網羅的に解析した。その結果、mir-155を含むいくつかのmicroRNAに有意差を認めており、実際に抗血小板薬を用いた次の研究段階に移る予定である。

LPSを用いた単球系細胞敗血症モデルにおいて、細胞死、貪食能低下が経時的に引き起こされ、高糖濃度環境下ではこれらが促進した。そのメカニズムとして小胞体ストレスの関与が示唆された。また、炎症消退脂質であるレゾルビンD2投与により細胞死、CHOP発現の抑制、貪食能の改善を認め、敗血症に対する薬物治療へ応用できる可能性があると考える。さらに、次世代シーケンサIon PGMシステムを用いたmicroRNAの網羅的解析を行い、予備実験段階ではあるがmiR-211,-204といったmicroRNAの変化を認め、今後更に敗血症におけるmicroRNAの関与についても検討していく予定である。

LPSを用いた単球系細胞敗血症モデルにおいて、高糖培養液下ではアポトーシスの促進および炎症性サイトカインの抑制が認められ、また貪食能の低下を認めた。そのメカニズムとして今回、小胞体ストレスおよびAktのリン酸化の関与が示唆された。また、炎症消退脂質として知られるレゾルビンD2を投与することによってアポトーシスの抑制、貪食能の改善を認め、今後の敗血症時の治療法として応用できる可能性があると考える。