Takahiro Sugiura

Total hydroperoxide and biological antioxidant potentials in a neonatal sepsis model.

Oxidant/antioxidant imbalance plays an important role in septic shock. The present study examined changes in circulating oxidative components in a neonatal sepsis model. Subjects were 14 newborn mixed-strain piglets randomly divided into two groups: a cecal ligation and perforation (CLP) model (n = 7) and sham (n = 7). Blood samples for total hydroperoxide (TH), biological antioxidant potential (BAP), tumor necrosis factor (TNF) α, interleukin (IL)-6, and IL-10 were collected pre-CLP and at 1, 3, and 6 h post-CLP. TH and BAP levels at 1 h post-CLP were significantly higher in the CLP group than in the sham group. In the CLP group, TH decreased gradually and reached baseline levels by 6 h post-CLP, while BAP remained elevated. Linear correlations were identified between serum TH and BAP at 1 h post-CLP, serum TH and TNF-α at 1 h post-CLP, and BAP and IL-6 at 6 h post-CLP. Changes in and correlations between circulating oxidative and inflammatory state components in a neonatal sepsis model were clarified. This is the first study to reveal that the presence of oxidant/antioxidant imbalance in sepsis and septic shock changes during the disease course.

Usefulness of a New Doppler Index for Assessing Both Ventricular Functions and Pulmonary Circulation in Newborn Piglet With Hypoxic Pulmonary Hypertension

Persistent pulmonary hypertension of the newborn is a clinical syndrome associated with a variety of cardiopulmonary diseases. Serial evaluation of pulmonary circulation and cardiac function is important, but available imaging techniques have been limited. A new Doppler index combining systolic and diastolic time intervals (the Tei index, which is a simple and noninvasive measurement) has been reported to be useful for the assessment of global cardiac function in adults and children. The purpose of this study was to test the effectiveness of the Tei index in prospectively assessing ventricular function and pulmonary circulation in a newborn piglet model with hypoxic pulmonary hypertension. One-day-old piglets (1.1–1.6 kg) were intubated and prepared for the experiments under room air and hypoxia. A complete two-dimensional Doppler echocardiographic examination was performed. Common hemodynamic variables were measured continuously throughout the study. The right ventricle (RV) Tei index under hypoxia (fraction of inspired oxygen = 0.10) was significantly higher than the value under air ventilation (medians, 0.38 versus 0.56;p < 0.05). Moreover, there was a significant correlation between RV Tei index and mean pulmonary artery pressure and positive linear correlation between individual changes in RV Tei index and changes in mPAP (r2 = 0.799, p < 0.05). We conclude that the Tei index is useful for assessing the function of the RV and the left ventricle and pulmonary circulation in a newborn piglet model with hypoxic pulmonary hypertension. These results suggest that the Tei index will become an objective method of assessing patients with persistent pulmonary hypertension of the newborn.

Edaravone, a novel free radical scavenger, reduces high-mobility group box 1 and prolongs survival in a neonatal sepsis model.

Free radicals play an important role in the inflammatory process of sepsis. We hypothesized that edaravone, a novel free radical scavenger, can suppress pathophysiological events and prolong survival in a neonatal sepsis cecal ligation and perforation (CLP) model. Of 32 3-day-old anesthetized and mechanically ventilated piglets, 11 received CLP only, 10 received CLP and edaravone treatment starting 30 min after CLP, and 11 constituted a sham (control) group. Mean arterial pressure (MAP), heart rate, cardiac output, arterial blood gas, serum total hydroperoxide, nitrite and nitrate, TNF-&agr;, and high-mobility group box 1 (HMGB1) were measured before CLP and at 1, 3, and 6 h after CLP. Compared with the CLP group, the edaravone group showed higher MAP at 6 h, lower heart rate at 1 and 3 h, lower total hydroperoxide at 1 h, lower nitrite and nitrate at 3 and 6 h, and higher (although not significantly so) mean cardiac output at 1, 3, and 6 h. TNF-&agr; elevation was delayed from 1 h in the CLP group to 3 h in the edaravone group. In the edaravone group, HMGB1 did not change significantly at any time, whereas in the CLP group, it increased at 6 h. Survival times were longer in the edaravone group than in the CLP group (15.4 ± 1.4 vs. 10.2 ± 1 h; P < 0.005). In addition, each of the serial dilutions of edaravone had a higher biological antioxidant potential than tempol does. In conclusion, edaravone suppressed free radicals, delayed the TNF-&agr; surge, and prevented HMGB1 elevation, thereby maintaining MAP and prolonging survival time in a neonatal sepsis CLP model.

Effect of hemoperfusion using polymyxin B-immobilized fiber on IL-6, HMGB-1, and IFN gamma in a neonatal sepsis model.

To evaluate effects of polymyxin B direct hemoperfusion (PMX-DHP) on a neonatal sepsis cecal ligation and perforation (CLP) model, in 24 anesthetized and mechanically ventilated 3-d-old piglets, 16 were assigned to CLP and an arteriovenous extracorporeal circuit from 3 h until 6 h post-CLP, with a PMX-column in PMX-DHP–treated group (8 piglets) and 8 as sham. Plasma lipopolysaccharide (LPS) was measured at before CLP and at 3 and 9 h. Changes in mean systemic blood pressure (mSBP), mean pulmonary blood pressure, serum IL-6, tumor necrosis factor alpha, interferon gamma, and highly mobile group-1 box protein were measured before CLP and at 1, 3, 6, and 9 h. LPS was lower in the sham and PMX-DHP groups than in the control at 9 h. The mSBP was higher in the sham and PMX-DHP groups than in the control at both 6 h. IL-6 was lower in the sham and PMX-DHP groups than in the control at 6 h. HMGB-1 was lower in the PMX-DHP group than in the control at 6 h. IFN-γ was only detected in the control group at 9 h. Survival times in the PMX-DHP group were longer than in the control. Thus, PMX-DHP improved septic shock in a neonatal septic model.

Development and characterization of a novel porcine model of neonatal sepsis.

Sepsis and its sequela remain a major source of morbidity and mortality in neonates despite advances in antimicrobials and aggressive supportive care. Many models of neonatal sepsis have been developed for investigating the pathophysiology of this disease and application of therapy, and a model with an infectious focus is closer to clinical reality. To establish an animal model that mimics the clinical characteristics of neonatal sepsis, the cecal devascularization and perforation procedure was implemented on 15 mixed-strain newborn piglets, which produced an infectious focus that acted as a continuous source of microorganisms to the peritoneal cavity. The mean survival time in animals with sepsis was 10.4 h (range 5.5-17.9 h), whereas all of the sham-operated control animals survived more than 24 h. Animals with sepsis showed a gradual significant decrease in the mean systemic blood pressure (mSBP; 71 ± 3 mmHg in sepsis vs. 64 ± 3 mmHg in control at 3 h, 38 ± 7 mmHg in sepsis vs. 59 ± 4 mmHg in control at 6 h, mean ± SEM). They also showed an increase of serum levels of endotoxin (5.6 × 105 ± 4.5 × 105 pg/mL in sepsis vs. 6.0 × 102 ± 3.8 × 102 pg/mL in control at 6 h). Serum levels of TNF-α in the animals with sepsis became significantly higher than the control animals at 0 h (96 ± 31 pg/mL in sepsis vs. 12 ± 1 pg/mL in control) and remained significantly higher than all through the experiment. Serum levels of IL-6 in animals with sepsis showed a gradual increase (484 ± 231 pg/mL in sepsis in its peak at 6 h vs. 24 ± 5 pg/mL in control), however, there were no significant differences in serum IL-10 levels between the groups. Microorganisms detected in the blood of animals with sepsis were gram-negative enteric and anaerobic organisms. These results suggested that this model mimics the clinical state of neonatal sepsis and hence may have significant implications for the treatment of sepsis, including its use as a model in further investigations.

A Method for Simultaneous Determination of 25-Hydroxyvitamin D3 and Its 3-Sulfate in Newborn Plasma by LC/ESI-MS/MS after Derivatization with a Proton-Affinitive Cookson-Type Reagent

A method for the simultaneous determination of 25-hydroxyvitamin D3 [25(OH)D3] and its 3-sulfate [25(OH)D3S] in newborn plasma, which is expected to be helpful in the assessment of the vitamin D status, using stable isotope-dilution liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) has been developed and validated. The plasma was pretreated based on the deproteinization and solid-phase extraction, then subjected to derivatization with 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD). The derivatization enabled the accurate quantification of 25(OH)D3 without interference from 3-epi-25(OH)D3 and also facilitated the simultaneous determination of the two metabolites by LC/positive ESI-MS/MS. Quantification was based on the selected reaction monitoring with the characteristic fragmentation of the DAPTAD-derivatives during MS/MS. This method was reproducible (intra- and inter-assay relative standard deviations of 7.8% or lower for both metabolites) and accurate (analytical recovery, 95.4-105.6%). The limits of quantification were 1.0 ng/mL and 2.5 ng/mL for 25(OH)D3 and 25(OH)D3S, respectively, when using a 20-μL sample. The developed method was applied to the simultaneous determination of plasma 25(OH)D3 and 25(OH)D3S in newborns; it was recognized that the plasma concentration of 25(OH)D3S is significantly higher than that of 25(OH)D3, and preterm newborns have lower plasma 25(OH)D3S concentrations than full-term newborns.

Endothelin receptor antagonist attenuates oxidative stress in a neonatal sepsis piglet model

Background:Oxidative stress (oxidant–antioxidant imbalance) plays an important role in the pathophysiology of neonatal sepsis. This study evaluated whether an antisense peptide endothelin receptor antagonist, ETR-P1/fl, could attenuate oxidative stress in a neonatal sepsis model.Methods:A total of 18 3-d-old piglets were anesthetized and mechanically ventilated. Six piglets received cecal ligation and perforation (CLP group) for induction of sepsis. Six piglets also received continuous infusion (0.05 mg/kg/h) of ETR-P1/fl 30 min after CLP (ETR-P1/fl group). Six piglets received a sham operation. Serum total hydroperoxide (TH), biological antioxidant potentials (BAPs), oxidative stress index (OSI, calculated as TH/BAP), interleukin (IL)-6, serum glutamic oxaloacetic transaminase (GOT), and creatinine were measured before CLP and at 1, 3, and 6 h after CLP.Results:CLP evoked a state of shock resulting in elevated TH, OSI, and IL-6 levels. ETR-P1/fl administration after CLP resulted in lower serum TH at 1 and 3 h after CLP, OSI at 1 and 3 h after CLP, IL-6 at 1 and 3 h after CLP, and GOT at 3 and 6 h after CLP as compared with the CLP group.Conclusion:ETR-P1/fl treatment significantly attenuated the elevation of serum oxidative stress markers (TH and OSI), IL-6, and GOT in a progressive neonatal sepsis CLP model.

The Tei index permits evaluation of cardiopulmonary function during inhaled nitric oxide therapy in the hypoxic newborn piglet.

The purpose of this study was to test the effectiveness of a new Doppler index combining systolic and diastolic time intervals (the Tei index) in the prospective assessment of ventricular function and pulmonary circulation in a newborn piglet model with hypoxic pulmonary hypertension during inhaled nitric oxide (NO). Piglets were prepared for the experiments under normal air, hypoxia, and hypoxia-with-inhaled-NO conditions. Complete two-dimensional Doppler echocardiographic examinations were performed in each condition. The right-ventricle (RV) Tei index increased with hypoxia and decreased following the inhalation of NO. Furthermore, there was a direct correlation between the individual changes in the RV Tei index and individual changes in mean pulmonary arterial pressure in each condition. We conclude that the Tei index is useful for assessing cardiac function and pulmonary circulation in hypoxic pulmonary hypertension during inhaled NO. These results suggest that the Tei index provides an objective assessment of persistent pulmonary hypertension of the newborn.

Enhancing analysis throughput, sensitivity and specificity in LC/ESI–MS/MS assay of plasma 25-hydroxyvitamin D3 by derivatization with triplex 4-(4-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) isotopologues

&NA; The plasma/serum concentration of 25‐hydroxyvitamin D3 [25(OH)D3] is a diagnostic index for vitamin D deficiency/insufficiency, which is associated with a wide range of diseases, such as rickets, cancer and diabetes. We have reported that the derivatization with 4‐(4‐dimethylaminophenyl)‐1,2,4‐triazoline‐3,5‐dione (DAPTAD) works well in the liquid chromatography/electrospray ionization‐tandem mass spectrometry (LC/ESI–MS/MS) assay of the serum/plasma 25(OH)D3 for enhancing the sensitivity and the separation from a potent interfering metabolite, 3‐epi‐25‐hydroxyvitamin D3 [3‐epi‐25(OH)D3]. However, enhancing the analysis throughput remains an issue in the LC/ESI–MS/MS assay of 25(OH)D3. The most obvious restriction of the LC/MS/MS throughput is the chromatographic run time. In this study, we developed an enhanced throughput method for the determination of the plasma 25(OH)D3 by LC/ESI–MS/MS combined with the derivatization using the triplex (2H0‐, 2H3‐ and 2H6‐) DAPTAD isotopologues. After separate derivatization with 1 of 3 different isotopologues, the 3 samples were combined and injected together into LC/ESI–MS/MS. Based on the mass differences between the isotopologues, the derivatized 25(OH)D3 in the 3 different samples were quantified within a single run. The developed method tripled the hourly analysis throughput without sacrificing assay performance, i.e., ease of pretreatment of plasma sample (only deproteinization), limit of quantification (1.0 ng/mL when a 5 &mgr;L‐plasma was used), precision (intra‐assay RSD ≤ 5.9% and inter‐assay RSD ≤ 5.5%), accuracy (98.7–102.2%), matrix effects, and capability of separating from an interfering metabolite, 3‐epi‐25(OH)D3. The multiplexing of samples by the isotopologue derivatization was applied to the analysis of plasma samples of healthy subjects and the developed method was proven to have a satisfactory applicability. HighlightsAn enhanced throughput LC/ESI–MS/MS method was developed for the 25(OH)D3 measurement.The method employed the derivatization with the triplex DAPTAD isotopologues.Use of isotopologues enabled the analyses of 3 different samples within a single run.The method was successfully applied to the analysis of adult and infant plasma samples.

Oxygen delivery using neonatal self-inflating bags without reservoirs.

Guidelines recommend avoiding excessive oxygen during neonatal resuscitation. Recent studies have suggested that oxygen titration can be achieved using a self‐inflating bag, but data on the effectiveness of resuscitators used in neonatal ventilation are scarce, The aim of this study was therefore to determine the amount of oxygen delivered using several brands of neonatal self‐inflating resuscitation bags without reservoirs under different conditions with regard to oxygen flow rate, ventilation rate (VR), peak inspiratory pressure (PIP) range, and test lung compliance.