Current Status and Associated Factors of Post-Hemorrhagic Hydrocephalus in Infants of 22 to 28 Weeks Gestation With Severe Intraventricular Hemorrhage in Korea: A Nationwide Cohort Study.

BACKGROUND: Post-hemorrhagic hydrocephalus (PHH), a common complication of severe intraventricular hemorrhage (IVH) in very low birth weight (BW) infants, is associated with significant morbidity and poor neurological outcomes. The objective of this study was to assess the current status of PHH and analyze the risk factors associated with the necessity of treatment for PHH in infants born between 22 and 28 weeks of gestation, specifically those with severe IVH (grade 3 or 4). METHODS: The analysis was conducted on 1,097 infants who were born between 22-28 gestational weeks and diagnosed with severe IVH, using data from the Korean Neonatal Network. We observed that the prevalence of PHH requiring treatment was 46.3% in infants with severe IVH. RESULTS: Higher rates of mortality, transfer during admission, cerebral palsy, and ventriculoperitoneal shunt after discharge were higher in infants with PHH than in those without PHH. PHH in severe IVH was associated with a higher rate of pulmonary hemorrhage, seizures, and IVH grade 4 in the entire cohort. In addition, it was associated with a lower rate of small for gestational age and chorioamnionitis. In the subgroup analysis, high BW, outborn status, pulmonary hemorrhage, seizure, sepsis, and IVH grade 4 were associated with a higher incidence of PHH between 22 and 25 gestational weeks (GW). In infants born between 26 and 28 GW, a higher incidence of PHH was associated with seizures and IVH grade 4. CONCLUSION: It is necessary to maintain meticulous monitoring and neurological intervention for infants with PHH not only during admission but also after discharge. In addition, identifying the clinical factors that increase the likelihood of developing PHH from severe IVH is crucial.

Diagnostic Utility of Whole Genome Sequencing After Negative Karyotyping/Chromosomal Microarray in Infants Born With Multiple Congenital Anomalies.

BACKGROUND: Achieving a definitive genetic diagnosis of unexplained multiple congenital anomalies (MCAs) in neonatal intensive care units (NICUs) infants is challenging because of the limited diagnostic capabilities of conventional genetic tests. Although the implementation of whole genome sequencing (WGS) has commenced for diagnosing MCAs, due to constraints in resources and faculty, many NICUs continue to utilize chromosomal microarray (CMA) and/or karyotyping as the initial diagnostic approach. We aimed to evaluate the diagnostic efficacy of WGS in infants with MCAs who have received negative results from karyotyping and/or CMA. METHODS: In this prospective study, we enrolled 80 infants with MCAs who were admitted to a NICU at a single center and had received negative results from CMA and/or karyotyping. The phenotypic characteristics were classified according to the International Classification of Diseases and the Human Phenotype Ontology. We assessed the diagnostic yield of trio-WGS in infants with normal chromosomal result and explored the process of diagnosing by analyzing both phenotype and genotype. Also, we compared the phenotype and clinical outcomes between the groups diagnosed with WGS and the undiagnosed group. RESULTS: The diagnostic yield of WGS was 26% (21/80), of which 76% were novel variants. There was a higher diagnostic yield in cases of craniofacial abnormalities, including those of the eye and ear, and a lower diagnostic yield in cases of gastrointestinal and genitourinary abnormalities. In addition, higher rates of rehabilitation therapy and gastrostomy were observed in WGS-diagnosed infants than in undiagnosed infants. CONCLUSION: This prospective cohort study assessed the usefulness of trio-WGS following chromosomal analysis for diagnosing MCAs in the NICU and revealed improvements in the diagnostic yield and clinical utility of WGS.

Five-year follow-up of phase II trial of stromal cells for bronchopulmonary dysplasia.

BACKGROUND: We previously performed a phase II randomised double-blind clinical trial of mesenchymal stromal cell (MSCs) transplantation to prevent bronchopulmonary dysplasia in extremely premature infants. Subsequently, we followed the infants enrolled in this clinical trial to determine the safety and effectiveness of MSCs against bronchopulmonary dysplasia at 5-year follow-up. METHODS: We evaluated infants at 5 years of age receiving placebo or MSCs in a prospective follow-up study. RESULTS: In terms of the primary end point of composite respiratory morbidities, including respiratory problem-related readmission, emergency department visits or oxygen therapy, the MSC group had a rate of 60.7% for composite morbidities, while the control group showed a tendency of higher rate of 83.9% for the same outcomes without statistical significance. In terms of the secondary outcomes, the MSC group infants showed a tendency of being less likely to visit emergency department (control 67.7% vs MSC 35.7%) and to receive oxygen therapy (control 29.0% vs MSC 3.6%). No difference was observed in the incidence of respiratory problem-related hospital readmission or wheezing episodes between the groups. CONCLUSION: Intratracheally instilled MSCs showed the possibility of potential to decrease respiratory symptom-related emergency department visits and oxygen therapy episodes in infants born extremely preterm during the 5 years after a phase II randomised controlled, double-blind trial of MSCs transplantation for bronchopulmonary dysplasia. This small size study suggests preliminary insights that can be further tested using larger sample sizes. TRIAL REGISTRATION NUMBER: NCT01897987.

Intratracheal Transplantation of Mesenchymal Stem Cells Attenuates Hyperoxia-Induced Microbial Dysbiosis in the Lungs, Brain, and Gut in Newborn Rats.

We attempted to determine whether intratracheal (IT) transplantation of mesenchymal stem cells (MSCs) could simultaneously attenuate hyperoxia-induced lung injuries and microbial dysbiosis of the lungs, brain, and gut in newborn rats. Newborn rats were exposed to hyperoxia (90% oxygen) for 14 days. Human umbilical cord blood-derived MSCs (5 × 105) were transplanted via the IT route on postnatal day (P) five. At P14, the lungs were harvested for histological, biochemical, and microbiome analyses. Bacterial 16S ribosomal RNA genes from the lungs, brain, and large intestine were amplified, pyrosequenced, and analyzed. IT transplantation of MSCs simultaneously attenuated hyperoxia-induced lung inflammation and the ensuing injuries, as well as the dysbiosis of the lungs, brain, and gut. In correlation analyses, lung interleukin-6 (IL-6) levels were significantly positively correlated with the abundance of Proteobacteria in the lungs, brain, and gut, and it was significantly inversely correlated with the abundance of Firmicutes in the gut and lungs and that of Bacteroidetes in the lungs. In conclusion, microbial dysbiosis in the lungs, brain, and gut does not cause but is caused by hyperoxic lung inflammation and ensuing injuries, and IT transplantation of MSCs attenuates dysbiosis in the lungs, brain, and gut, primarily by their anti-oxidative and anti-inflammatory effects.

Mesenchymal Stem Cells and Formyl Peptide Receptor 2 Activity in Hyperoxia-Induced Lung Injury in Newborn Mice.

Formyl peptide receptor (FPR) 2 is known to play a critical role in regulating inflammation, including either the pro-inflammatory or pro-resolving effects. However, its role in neonatal hyperoxia-induced lung injury has not been delineated. In this study, we investigate whether mesenchymal stem cells (MSCs) attenuate hyperoxia-induced neonatal lung injury by regulating FPR2 activity. We observed a significant increase in FPR2 levels in alveolar macrophages (RAW264.7 cells) after H2O2-induced stress, which decreased after MSC treatment. In the H2O2-induction model, increased levels of inflammatory cytokines (IL-1α and TNF-α) were significantly reduced in RAW264.7 cells after treatment with WRW4, an inhibitor of FPR2, or MSCs. Viability of lung epithelial cells and endothelial cells was significantly improved when cultured in the conditioned media of RAW264.7 cells treated with WRW4 or MSCs, compared to when cultured in the conditioned media of control RAW265.7 cells exposed to H2O2. For the in vivo study, wild-type and FPR2 knockout (FPR2-/-) C57/BL6 mouse pups were randomly exposed to 80% oxygen or room air from postnatal day (P) 1 to P14. At P5, 2 × 105 MSCs were transplanted intratracheally. MSCs reduced the elevated FPR2 activity at P7 and improved the decreased FPR2 activity as well as the increased immuno-stained FPR2 activity in alveolar macrophages in hyperoxic lungs at P14. Both FPR2-/- and MSCs similarly attenuated impaired alveolarization and angiogenesis, and increased apoptosis and inflammation of hyperoxic lungs without synergistic effects. Our findings suggest that the protective effects of MSCs in hyperoxic lung injury might be related to indirect modulation of FPR2 activity, at least of alveolar macrophages in neonatal mice.

Thrombin Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage Induced Neonatal Rats.

Severe intraventricular hemorrhage (IVH) remains a major cause of high mortality and morbidity in extremely preterm infants. Mesenchymal stem cell (MSC) transplantation is a possible therapeutic option, and development of therapeutics with enhanced efficacy is necessary. This study investigated whether thrombin preconditioning improves the therapeutic efficacy of human Wharton's jelly-derived MSC transplantation for severe neonatal IVH, using a rat model. Severe neonatal IVH was induced by injecting 150 µL blood into each lateral ventricle on postnatal day (P) 4 in Sprague-Dawley rats. After 2 days (P6), naïve MSCs or thrombin-preconditioned MSCs (1 × 105/10 µL) were transplanted intraventricularly. After behavioral tests, brain tissues and cerebrospinal fluid of P35 rats were obtained for histological and biochemical analyses, respectively. Thrombin-preconditioned MSC transplantation significantly reduced IVH-induced ventricular dilatation on in vivo magnetic resonance imaging, which was coincident with attenuations of reactive gliosis, cell death, and the number of activated microglia and levels of inflammatory cytokines after IVH induction, compared to naïve MSC transplantation. In the behavioral tests, the sensorimotor and memory functions significantly improved after transplantation of thrombin-preconditioned MSCs, compared to naïve MSCs. Overall, thrombin preconditioning significantly improves the therapeutic potential and more effectively attenuates brain injury, including progressive ventricular dilatation, gliosis, cell death, inflammation, and neurobehavioral functional impairment, in newborn rats with induced severe IVH than does naïve MSC transplantation.

Mortality and Morbidities according to Time of Birth in Extremely Low Birth Weight Infants.

BACKGROUND: Although the overall quality of high-risk neonatal care has improved recently, there is still concern about a difference in the quality of care when comparing off-hour births and regular-hour births. Moreover, there are no data in Korea regarding the impact of time of birth on mortality and morbidities in preterm infants. METHODS: A total of 3,220 infants weighing < 1,000 g and born at 23-34 weeks in 2013-2017 were analyzed based on the Korean Neonatal Network data. Mortality and major morbidities were analyzed using logistic regression according to time of birth during off-hours (nighttime, weekend, and holiday) and regular hours. The institutes were sub-grouped into hospital group I and hospital group II based on the neonatal intensive care unit (NICU) care level defined by the mortality rates of < 50% and ≥ 50%, respectively, in infants born at 23-24 weeks' gestation. RESULTS: The number of births during regular hours and off-hours was similar. In the total population and hospital group I, off-hour births were not associated with increased neonatal mortality and morbidities. However, in hospital group II, increased early mortality was found in the off-hour births when compared to regular-hour births. CONCLUSION: Efforts to improve the overall quality of NICU are required to lower the early mortality rate in off-hour births. Also, other sensitive indexes for the evaluation of quality of NICU care should be further studied.

BDNF-Overexpressing Engineered Mesenchymal Stem Cells Enhances Their Therapeutic Efficacy against Severe Neonatal Hypoxic Ischemic Brain Injury.

We investigated whether irradiated brain-derived neurotropic factor (BDNF)-overexpressing engineered human mesenchymal stem cells (BDNF-eMSCs) improve paracrine efficiency and, thus, the beneficial potency of naïve MSCs against severe hypoxic ischemic (HI) brain injury in newborn rats. Irradiated BDNF-eMSCs hyper-secreted BDNF > 10 fold and were >5 fold more effective than naïve MSCs in attenuating the oxygen-glucose deprivation-induced increase in cytotoxicity, oxidative stress, and cell death in vitro. Only the irradiated BDNF-eMSCs, but not naïve MSCs, showed significant attenuating effects on severe neonatal HI-induced short-term brain injury scores, long-term progress of brain infarct, increased apoptotic cell death, astrogliosis and inflammatory responses, and impaired negative geotaxis and rotarod tests in vivo. Our data, showing better paracrine potency and the resultant better therapeutic efficacy of the irradiated BDNF-eMSCs, compared to naïve MSCs, suggest that MSCs transfected with the BDNF gene might represent a better, new therapeutic strategy against severe neonatal HI brain injury.

Reactive microglia and astrocytes in neonatal intraventricular hemorrhage model are blocked by mesenchymal stem cells.

Severe intraventricular hemorrhage (IVH) in premature infants triggers reactive gliosis, causing acute neuronal death and glial scar formation. Transplantation of mesenchymal stem cells (MSCs) has often showed improved CNS recovery in an IVH model, but whether this response is related to reactive glial cells is still unclear. Herein, we suggest that MSCs impede the response of reactive microglia rather than astrocytes, thereby blocking neuronal damage. Astrocytes alone showed mild reactiveness under hemorrhagic conditions mimicked by thrombin treatment, and this was not blocked by MSC-conditioned medium (MSC-CM) in vitro. In contrast, thrombin-induced microglial activation and release of proinflammatory cytokines were inhibited by MSC-CM. Interestingly, astrocytes showed greater reactive response when co-cultured with microglia, and this was abolished in the presence of MSC-CM. Gene expression profiles in microglia revealed that transcript levels of genes for immune response and proinflammatory cytokines were altered by thrombin treatment. This result coincided with the robust phosphorylation of STAT1 and p38 MAPK, which might be responsible for the production and release of proinflammatory cytokines. Furthermore, application of MSC-CM diminished thrombin-mediated phosphorylation of STAT1 and p38 MAPK, supporting the acute anti-inflammatory role of MSCs under hemorrhagic conditions. In line with this, activation of microglia and consequent cytokine release were impaired in Stat1-null mice. However, reactive response in Stat1-deficient astrocytes was maintained. Taken together, our results demonstrate that MSCs mainly block the activation of microglia involving STAT1-mediated cytokine release and subsequent reduction of reactive astrocytes.

Developing a newborn rat model of ventriculitis without concomitant bacteremia by intraventricular injection of K1 (-) Escherichia coli.

BACKGROUND: Neonatal meningitis caused by Escherichia coli results in high mortality and neurological disabilities, and the concomitant systemic bacteremia confounds its mortality and brain injury. This study developed an experimental model of neonatal ventriculitis without concomitant systemic bacteremia by determining the bacterial inoculum of K1 capsule-negative E. coli by intraventricular injection in newborn rats. METHODS: We carried out intraventricular injections 1 × 102 (low dose), 5 × 102 (medium dose), or 1 × 103 (high dose) colony-forming units (CFU) of K1 (-) E. coli (EC5ME) in Sprague-Dawley rats at postnatal day (P) 11. Ampicillin was started at P12. Blood and cerebrospinal fluid (CSF) cultures were performed at 6 h, 1 day, and 6 days after inoculation. Brain magnetic resonance imaging (MRI) was performed at P12 and P17. Survival was monitored, and brain tissue was obtained for histological and biochemical analyses at P12 and P17. RESULTS: Survival was inoculum dose-dependent, with the lowest survival in the high-dose group (20%) compared with the medium- (67%) or low- (73%) dose groups. CSF bacterial counts in the low- and medium-dose groups were significantly lower than that in the high-dose group at 6 h, but not at 24 h after inoculation. No bacteria were isolated from the blood throughout the experiment or from the CSF at P17. Brain MRI showed an inoculum dose-dependent increase in the extent of brain injury and inflammatory responses. CONCLUSIONS: We developed a newborn rat model of bacterial ventriculitis without concomitant systemic bacteremia by intraventricular injection of EC5ME.

B23/Nucleophosmin promotes reconstitution of synaptic path in hippocampus after injury.

B23, also known as nucleophosmin (NPM), is multifunctional protein directly implicated in cell proliferation, cell cycle progression, and cell survival. In the current study, in addition to confirming its anti-apoptotic function in neuronal survival, we demonstrated that the spatial-temporal expression profile of B23 during development of hippocampal neurons is high in the embryonic stage, down-regulated after birth, and preferentially localized at the tips of growing neuritis and branching points. Overexpression of B23 promotes axon growth with abundant branching points in growing hippocampal neurons, but depletion of B23 impairs axon growth, leading to neuronal death. Following injury to the trisynaptic path in hippocampal slice, overexpression of B23 remarkably increased the number and length of regenerative fibers in the mossy fiber path. Our study suggests that B23 expression in developing neurons is essential for neuritogenesis and axon growth and that up-regulation of B23 may be a strategy for enhancing the reconstitution of synaptic paths after injury to hippocampal synapses.

Outcome and risk factors associated with perirenal subcapsular fluid collections in extremely preterm infants with acute kidney injury.

OBJECTIVES: To investigate the incidence of, clinical outcome of, and risk factors for perirenal subcapsular fluid collections in extremely preterm infants with acute kidney injury (AKI). METHODS: Extremely preterm infants with AKI who underwent renal ultrasonography (US) during neonatal intensive care unit stay were classified into two groups according to the presence of a perirenal subcapsular fluid collection at US. Clinical outcome was compared, and relevant data were analysed, including demographics and comorbidities of the infants, as well as maternal demographics. The risk factor of perirenal subcapsular fluid in infants with AKI was tested with univariate and multivariate logistic regression analysis. RESULTS: A perirenal subcapsular fluid collection was detected in 7 of 56 (13%) extremely preterm infants with AKI (male to female ratio, 5:2; mean gestational age, 23.6 ± 1.4 weeks) and it appeared bilaterally in most cases (86%, 6/7). The mortality rate was higher in infants with perirenal subcapsular fluid collections and AKI (86%, 6/7) than with AKI alone (35%, 17/49) (p = 0.015). Infants with perirenal subcapsular fluid collections and AKI were of a lower gestational age, and more frequently showed episodes of intestinal perforation, use of medication having potential to impair renal function, and a history of maternal chorioamnionitis (p < 0.05). Multivariate analysis revealed a significantly higher risk for perirenal subcapsular fluid collections in extremely preterm infants who were treated with anti-fungal agents (OR, 13.2 (95% CI: 1.5, 119.4); p = 0.022). CONCLUSIONS: Although a perirenal subcapsular fluid collection occurred in a small proportion of extremely preterm infants with AKI, its presence was associated with high mortality. The use of anti-fungal agents was an independent risk factor for a perirenal subcapsular fluid collection. KEY POINTS: • A perirenal subcapsular fluid collection may occur in association with acute kidney injury. • A perirenal subcapsular fluid collection has a grave prognostic implication in extremely preterm infants. • The use of anti-fungal agent might be associated with perirenal subscapular fluid collections in critically ill extremely preterm infants with AKI.

Mortality Rate-Dependent Variations in the Timing and Causes of Death in Extremely Preterm Infants Born at 23-24 Weeks' Gestation.

OBJECTIVES: To determine mortality rate-dependent variations in the timing and causes of death, and to subsequently identify the clinical factors associated with decreased mortality in extremely preterm infants born at 23-24 weeks' gestation. DESIGN: A retrospective cohort study. SETTING: Korean Neonatal Network registry that includes all level greater than or equal to 3 neonatal ICUs in Korea. PATIENTS: Eligible, actively treated infants born at 23-24 weeks' gestation (n = 574) from January 2014 to December 2016 were arbitrarily categorized based on institutional mortality rates of less than or equal to 50% (group I, n = 381) and greater than 50% (group II, n = 193). The primary outcome was mortality before discharge and the timing and causes of death according to the mortality rate. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The overall mortality rate was significantly lower in group I (40.7%) than in group II (79.3%). Regarding causes of death, mortalities due to cardiorespiratory, infectious, and gastrointestinal causes were significantly lower in group I than in group II. Mortality rates were significantly lower in group I, including all the subgroups that were categorized according to the timing of death, than in group II. The multivariate analyses showed that antenatal corticosteroid use, absence of oligohydramnios, birth weight, and body temperature at admission to the neonatal ICU were significantly associated with reduced mortality. CONCLUSIONS: The reduced mortality rate among the infants born at 23-24 weeks' gestation was attributable to decreased mortality ascribed to cardiorespiratory, infectious, and gastrointestinal causes, and it was associated with antenatal steroid use and body temperature at admission to the neonatal ICU.

Initial and delayed thyroid-stimulating hormone elevation in extremely low-birth-weight infants.

BACKGROUND: To determine the incidence, etiology, and outcomes of thyroid-stimulating hormone (TSH) elevation in extremely low-birth-weight infants (ELBWIs). METHODS: Newborn thyroid screening data of 584 ELBWIs (birth weight, < 1000 g; gestational age, ≥ 23 weeks) were retrospectively analyzed to identify initial (≤ 2 postnatal weeks) and delayed (> 2 weeks) TSH elevations. Growth and neurodevelopmental outcomes at 2 years' corrected age (CA) were assessed according to levothyroxine replacement. RESULTS: Initial and delayed TSH elevations were detected at CAs of 27 and 30 weeks, respectively, with incidence rates of 0.9 and 7.2%, respectively. All infants with initial TSH elevations had perinatal asphyxia, and 95% of those with delayed TSH elevation were exposed to various stressors, including respiratory support, drugs, and surgery within 2 weeks before diagnosis of TSH elevation. Free thyroxine (T4) levels were simultaneously reduced in 80 and 57% of infants with initial and delayed TSH elevations, respectively. Both initial and delayed TSH elevations were transient, regardless of levothyroxine replacement. Infants receiving levothyroxine replacement therapy had significantly higher TSH elevations, significantly lower free T4 levels, and significantly reduced mortality, compared to untreated infants. However, levothyroxine replacement had no significant effect on long-term growth and neurodevelopmental outcomes. CONCLUSIONS: The timing of insult superimposition on hypothalamic-pituitary-thyroid axis maturation is a major determinant of initial or delayed TSH elevation in ELBWIs. Levothyroxine replacement did not affect growth or neurodevelopmental outcomes in this population.

The Impact of Surgical Intervention on Neurodevelopmental Outcomes in Very Low Birth Weight Infants: a Nationwide Cohort Study in Korea.

BACKGROUND: To investigate the incidence of surgical intervention in very low birth weight (VLBW) infants and the impact of surgery on neurodevelopmental outcomes at corrected ages (CAs) of 18-24 months, using data from the Korean Neonatal Network (KNN). METHODS: Data from 7,885 VLBW infants who were born and registered with the KNN between 2013 to 2016 were analyzed in this study. The incidences of various surgical interventions and related morbidities were analyzed. Long-term neurodevelopmental outcomes at CAs of 18-24 months were compared between infants (born during 2013 to 2015, n = 3,777) with and without surgery. RESULTS: A total of 1,509 out of 7,885 (19.1%) infants received surgical interventions during neonatal intensive care unit (NICU) hospitalization. Surgical ligation of patent ductus arteriosus (n = 840) was most frequently performed, followed by laser therapy for retinopathy of prematurity and laparotomy due to intestinal perforation. Infants who underwent surgery had higher mortality rates and greater neurodevelopmental impairment than infants who did not undergo surgery (P value < 0.01, both). On multivariate analysis, single or multiple surgeries increased the risk of neurodevelopmental impairment compared to no surgery with adjusted odds ratios (ORs) of 1.6 with 95% confidence interval (CI) of 1.1-2.6 and 2.3 with 95% CI of 1.1-4.9. CONCLUSION: Approximately one fifth of VLBW infants underwent one or more surgical interventions during NICU hospitalization. The impact of surgical intervention on long-term neurodevelopmental outcomes was sustained over a follow-up of CA 18-24 months. Infants with multiple surgeries had an increased risk of neurodevelopmental impairment compared to infants with single surgeries or no surgeries after adjustment for possible confounders.

Effect of Sustained Inflations vs Intermittent Positive Pressure Ventilation on Bronchopulmonary Dysplasia or Death Among Extremely Preterm Infants: The SAIL Randomized Clinical Trial.

Importance: Preterm infants must establish regular respirations at delivery. Sustained inflations may establish lung volume faster than short inflations. Objective: To determine whether a ventilation strategy including sustained inflations, compared with standard intermittent positive pressure ventilation, reduces bronchopulmonary dysplasia (BPD) or death at 36 weeks' postmenstrual age without harm in extremely preterm infants. Design, Setting, and Participants: Unmasked, randomized clinical trial (August 2014 to September 2017, with follow-up to February 15, 2018) conducted in 18 neonatal intensive care units in 9 countries. Preterm infants 23 to 26 weeks' gestational age requiring resuscitation with inadequate respiratory effort or bradycardia were enrolled. Planned enrollment was 600 infants. The trial was stopped after enrolling 426 infants, following a prespecified review of adverse outcomes. Interventions: The experimental intervention was up to 2 sustained inflations at maximal peak pressure of 25 cm H2O for 15 seconds using a T-piece and mask (n = 215); standard resuscitation was intermittent positive pressure ventilation (n = 211). Main Outcome and Measures: The primary outcome was the rate of BPD or death at 36 weeks' postmenstrual age. There were 27 prespecified secondary efficacy outcomes and 7 safety outcomes, including death at less than 48 hours. Results: Among 460 infants randomized (mean [SD] gestational age, 25.30 [0.97] weeks; 50.2% female), 426 infants (92.6%) completed the trial. In the sustained inflation group, 137 infants (63.7%) died or survived with BPD vs 125 infants (59.2%) in the standard resuscitation group (adjusted risk difference [aRD], 4.7% [95% CI, -3.8% to 13.1%]; P = .29). Death at less than 48 hours of age occurred in 16 infants (7.4%) in the sustained inflation group vs 3 infants (1.4%) in the standard resuscitation group (aRD, 5.6% [95% CI, 2.1% to 9.1%]; P = .002). Blinded adjudication detected an imbalance of rates of early death possibly attributable to resuscitation (sustained inflation: 11/16; standard resuscitation: 1/3). Of 27 secondary efficacy outcomes assessed by 36 weeks' postmenstrual age, 26 showed no significant difference between groups. Conclusions and Relevance: Among extremely preterm infants requiring resuscitation at birth, a ventilation strategy involving 2 sustained inflations, compared with standard intermittent positive pressure ventilation, did not reduce the risk of BPD or death at 36 weeks' postmenstrual age. These findings do not support the use of ventilation with sustained inflations among extremely preterm infants, although early termination of the trial limits definitive conclusions. Trial Registration: clinicaltrials.gov Identifier: NCT02139800.

Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways.

We investigated the role of protease-activated receptor (PAR)-mediated signaling pathways in the biogenesis of human umbilical cord blood-derived mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) and the enrichment of their cargo content after thrombin preconditioning. Immunoblot analyses showed that MSCs expressed two PAR subtypes: PAR-1 and PAR-3. Thrombin preconditioning significantly accelerated MSC-derived EV biogenesis more than five-fold and enriched their cargo contents by more than two-fold via activation of Rab5, early endosomal antigen (EEA)-1, and the extracellular signal regulated kinase (ERK)1/2 and AKT signaling pathways. Blockage of PAR-1 with the PAR-1-specific antagonist, SCH79797, significantly suppressed the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways and subsequently increased EV production and enriched EV cargo contents. Combined blockage of PAR-1 and PAR-3 further and significantly inhibited the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways, accelerated EV production, and enriched EV cargo contents. In summary, thrombin preconditioning boosted the biogenesis of MSC-derived EVs and enriched their cargo contents largely via PAR-1-mediated pathways and partly via PAR-1-independent, PAR-3-mediated activation of Rab5, EEA-1, and the ERK1/2 and AKT signaling pathways.

Thrombin Preconditioning Enhances Therapeutic Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells in Severe Neonatal Hypoxic Ischemic Encephalopathy.

We investigated whether thrombin preconditioning of human Wharton's jelly-derived mesenchymal stem cells (MSCs) improves paracrine potency and thus the therapeutic efficacy of naïve MSCs against severe hypoxic ischemic encephalopathy (HIE). Thrombin preconditioning significantly enhances the neuroprotective anti-oxidative, anti-apoptotic, and anti-cytotoxic effects of naïve MSCs against oxygen-glucose deprivation (OGD) of cortical neurons in vitro. Severe HIE was induced in vivo using unilateral carotid artery ligation and hypoxia for 2 h and confirmed using brain magnetic resonance imaging (MRI) involving >40% of ipsilateral hemisphere at postnatal day (P) 7 in newborn rats. Delayed intraventricular transplantation of 1 × 105 thrombin preconditioned but not naïve MSCs at 24 h after hypothermia significantly enhanced observed anti-inflammatory, anti-astroglial, and anti-apoptotic effects and the ensuing brain infarction; behavioral tests, such as cylinder rearing and negative geotaxis tests, were conducted at P42. In summary, thrombin preconditioning of human Wharton's jelly-derived MSCs significantly boosted the neuroprotective effects of naïve MSCs against OGD in vitro by enhancing their anti-oxidative, anti-apoptotic, and anti-cytotoxic effects, and significantly attenuated the severe HIE-induced brain infarction and improved behavioral function tests in vivo by maximizing their paracrine anti-inflammatory, anti-astroglial, and anti-apoptotic effects.

Strategies to enhance paracrine potency of transplanted mesenchymal stem cells in intractable neonatal disorders.

Mesenchymal stem cell (MSC) transplantation represents the next breakthrough in the treatment of currently intractable and devastating neonatal disorders with complex multifactorial etiologies, including bronchopulmonary dysplasia, hypoxic ischemic encephalopathy, and intraventricular hemorrhage. Absent engraftment and direct differentiation of transplanted MSCs, and the "hit-and-run" therapeutic effects of these MSCs suggest that their pleiotropic protection might be attributable to paracrine activity via the secretion of various biologic factors rather than to regenerative activity. The transplanted MSCs, therefore, exert their therapeutic effects not by acting as "stem cells," but rather by acting as "paracrine factors factory." The MSCs sense the microenvironment of the injury site and secrete various paracrine factors that serve several reparative functions, including antiapoptotic, anti-inflammatory, antioxidative, antifibrotic, and/or antibacterial effects in response to environmental cues to enhance regeneration of the damaged tissue. Therefore, the therapeutic efficacy of MSCs might be dependent on their paracrine potency. In this review, we focus on recent investigations that elucidate the specifically regulated paracrine mechanisms of MSCs by injury type and discuss potential strategies to enhance paracrine potency, and thus therapeutic efficacy, of transplanted MSCs, including determining the appropriate source and preconditioning strategy for MSCs and the route and timing of their administration.

Mesenchymal stem cells transplantation attenuates brain injury and enhances bacterial clearance in Escherichia coli meningitis in newborn rats.

OBJECTIVE: Neonatal meningitis caused by Escherichia coli results in significant mortality and neurological disabilities, with few effective treatments. Recently, we demonstrated that human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) transplantation attenuated E. coli-induced severe pneumonia, primarily by reducing inflammation and enhancing bacterial clearance. This study aimed to determine whether intraventricular transplantation of hUCB-MSCs attenuated the brain injury in E. coli meningitis in newborn rats. METHODS: Meningitis without concomitant bacteremia was induced by intraventricular injection of 5 × 102 colony forming units of K1 (-) E. coli in rats at postnatal day (P)11, and hUCB-MSCs (1 × 105) were transplanted intraventricularly 6 h after induction of meningitis. Antibiotics was started 24 h after modeling. RESULT: Meningitis modeling induced robust proliferation of E. coli in the cerebrospinal fluid and increased mortality in rat pups, and MSC transplantation significantly reduced this bacterial growth and the mortality rate. Impaired sensorimotor function in the meningitis rats was ameliorated by MSCs injection. MSCs transplantation also attenuated meningitis caused brain injury including cerebral ventricular dilatation, brain cell death, reactive gliosis, and inflammatory response. CONCLUSION: Intraventricular transplantation of hUCB-MSCs significantly improved survival and attenuated the brain injury via anti-inflammatory and antibacterial effects in experimental neonatal E. coli meningitis.