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Objective Point-of-care ultrasound (POC US) has been increasingly used by intensive care physicians. Growing use of POC US necessitates defining distinct clinical indications for its application, as well as structured POC US training programs. Homogeneous approach to POC US education combined with rigorous quality assurance should further enable POC US to become standard-of-care clinical tool. This study aimed to present the first, innovative, and structured POC US program in neonatal–perinatal medicine field. In addition, we reviewed the availability of the POC US training programs across different medical specialties. Study Design Available English-language publications on POC US training programs in general and neonatal–perinatal medicine were reviewed in this study. Discussion Mounting body of evidence suggests improved procedural completion rates, as well as clinical decision making with the use of POC US. However, limited research supported the existence of structured, comprehensive POC US programs. It was recognized that medical institutions need to develop syllabuses, teach, and credential increasing number of health care professionals in the use of POC US. We defined intuitive educational strategy that encompasses POC US clinical indications, educational curriculum, scanning protocols, competence evaluation, and finally credentialing process. In addition, we offered description of the imaging quality assurance, as well as POC US coding, and reimbursement. Conclusion Future efforts need to be dedicated to the ongoing development of neonatal POC US as a clinical instrument. It should allow for eventual paradigm change and improved effectiveness in management of critically ill neonates.

Pharmacologic interventions to halt/reverse the vascular remodeling and right ventricular dysfunction in pulmonary arterial hypertension (PAH) remains an unmet need. We previously demonstrated extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a DAMP (damage-associated molecular pattern protein) contributing to PAH pathobiology via TLR4 ligation. We examined the role of endothelial cell (EC)-specific eNAMPT in experimental PH and an eNAMPT-neutralizing mAb as a therapeutic strategy to reverse established PH. Hemodynamic/echocardiographic measurements and tissue analyses were performed in Sprague Dawley rats exposed to 10% hypoxia/Sugen (three weeks) followed by return to normoxia and weekly intraperitoneal delivery of the eNAMPT mAb (1 mg/kg). WT C57BL/6J mice and conditional EC-cNAMPT ec−/− mice were exposed to 10% hypoxia (three weeks). Biochemical and RNA sequencing studies were performed on rat PH lung tissues and human PAH PBMCs. Hypoxia/Sugen-exposed rats exhibited multiple indices of severe PH (right ventricular systolic pressure, Fulton index), including severe vascular remodeling, compared to control rats. PH severity indices and plasma levels of eNAMPT, IL-6, and TNF- α were all significantly attenuated by eNAMPT mAb neutralization. Compared to hypoxia-exposed WT mice, cNAMPT ec−/− KO mice exhibited significantly reduced PH severity and evidence of EC to mesenchymal transition (EndMT). Finally, biochemical and RNAseq analyses revealed eNAMPT mAb-mediated rectification of dysregulated inflammatory signaling pathways (TLR/NF-κB, MAP kinase, Akt/mTOR) and EndMT in rat PH lung tissues and human PAH PBMCs. These studies underscore EC-derived eNAMPT as a key contributor to PAH pathobiology and support the eNAMPT/TLR4 inflammatory pathway as a highly druggable therapeutic target to reduce PH severity and reverse PAH.

Highlights • Persistent cloaca with caudal duplication and a vestigial appendage is very rare. • Initial management include decompressive colostomy and drainage of the hydrocolpos. • Staged surgery include repair of cloaca and removal of accessory foot. • Preserving quality of life and managing complications is of key importance., Introduction A cloaca occurs when genitourinary tract and bowel converge into a common channel. We report a case of partial caudal duplication, persistent cloaca and vestigial appendage in a monovular female twin infant. Presentation of case This is a monochorinonic-diamniotic twin born at 36 weeks with apgars of 9/9. She had a duplicated labia with two clitorises, and a partially formed accessory foot with 2 toes protruding from the right gluteal region. There was anal atresia and a punctate urethral opening in the right genitalia through which she voided spontaneously. X-ray of the accessory foot had rudimentary metatarsals and phalanges. There was left hydroureteronephrosis and a hydrocolpos causing severe mass effect. On the first day of life, she had exploratory laparotomy with a diverting colostomy and mucus fistula and drainage of hydrocolpos. At 6 months of age, she had removal of the accessory foot with flap closure of the perineal defect and vesicostomy. At 15 months of age she had laparotomy for repair of cloaca, excision of presacral pelvic mass and the duplicated vulva. Discussion Theories of etiology include failure of regression of Kovalevsky’s canal (a communication that connects the amniotic and yolk sac), an incomplete form of twinning through iatrogenic damage to the zona pellucida or a failed triplet formation from a single embryo. Conclusion Caudal duplication with persistent cloaca and vestigial appendage is a rare and complex malformation. Having a unified surgical and medical team to preserve quality of life and to treat complications is of key importance.

Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used anin-vitromodel: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study thein-vitromodel, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p< 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p< 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p< 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p< 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

BackgroundPeriventricular leukomalacia (PVL), a devastating brain injury affecting premature infants, is the most common cause of cerebral palsy. PVL is caused by hypoxia ischemia (HI) and is characterized by white matter necrotic lesions, microglial activation, upregulation of NF-κB, and neuronal death. The microglia is the main cell involved in PVL pathogenesis. The goal of this study was to investigate the role of microglial NF-κB activity and its prophylactic inhibition in a neonate mouse model of HI.MethodsTransgenic mice with specific knockout NF-κB in microglia and colony stimulating factor 1 receptor Cre with floxed IKKβ (CSF-1R Cre + IKKβflox/wt) were used. Postnatal day 5 (P5) mice underwent sham or bilateral temporary carotid artery ligation followed by hypoxia. After HI insult, inflammatory cytokines, volumetric MRI, histopathology, and immunohistochemistry for oligodendroglia and microglial activation markers were analyzed. Long-term neurobehavioral assessment, including grip strength, rotarod, and open field testing, was performed at P60.ResultsWe demonstrate that selective inhibition of NF-κB in microglia decreases HI-induced brain injury by decreasing microglial activation, proinflammatory cytokines, and nitrative stress. Rescue of oligodendroglia is evidenced by immunohistochemistry, decreased ventriculomegaly on MRI, and histopathology. This selective inhibition leads to attenuation of paresis, incoordination, and improved grip strength, gait, and locomotion.ConclusionWe conclude that NF-κb activation in microglia plays a major role in the pathogenesis of hypoxic ischemic injury of the immature brain, and its prophylactic inhibition offers significant neuroprotection. Using a specific inhibitor of microglial NF-κB may offer a new prophylactic or therapeutic alternative in preterm infants affected by HI and possibly other neurological diseases in which microglial activation plays a role.

The primary objective was to study agreement between X-rays and point of care ultrasound (POC-US) in determining central venous line (CVL) tip position. The secondary objective was to examine malposition rates over time using POC-US. Fifty-six neonates were enrolled who had a CVL placed. Initial X-rays and POC-US were obtained. POC-US was performed daily thereafter for the total of 6 days. US video clips were acquired in four standard echocardiographic views: subcostal, four-chamber, and short- and long-axis parasternal views. Gwet's agreement coefficient (AC1) for agreement measured inter-rater reliability of X-rays and POC-US (correct position/malposition). A generalized linear mixed model for binary clustered data estimated malposition rate over time. All analyses were conducted using SAS version 9.4 and Agree Stat. The study included 108 "pairs" of X-rays and POC-US images. Agreement coefficient (AC1), with respect to correct position/malposition of CVL tip, was high AC1 = 0.872 (UVC-AC1 = 0.814, PICC-AC1 = 0.94). Among birth weight (BW) 1000 g, 1000-1499 g, and BW 1500 g, AC1 values were 0.922, 0.774, and 0.873, respectively. CVL tip malposition rate decreased over time.Conclusions: Agreement between POC-US and X-rays for CVL tip position was high, with the highest in BW 1000 g. The data suggest that POC-US can be used for initial confirmation and follow up of CVL tip position. What is Known • X-ray is currently the gold standard for localizing central venous line (CVL) tip position. • Malposition of CVL tip can lead to life-threatening complications. What is New • POC-US is superior to X-ray as it can follow CVL tip position over time, detecting malpositioned lines, adjusting them in a timely manner thus preventing complications. • Standardizing CVL placement, X-ray acquisition, POC-US acquisition with four views with video clips and ultrasound operator training increases accuracy and thus agreement between X-ray and POC-US. • UVC tip is more likely to be malpositoned than PICC tip. Malposition of UVC tip using POC-US decreased over time due to shrinking of the umbilical cord in the first 48 of life.

Background Platelet and packed red blood cell (pRBC) transfusion practices for preterm infants vary significantly worldwide. Recent randomized trials of preterm infants receiving platelet or pRBC transfusions have not shown any benefit with higher compared to lower transfusion thresholds. In the recent PlaNeT2 study published in January 2019, infants receiving platelets at higher thresholds had significantly higher rates of death and major bleeding. These findings raise concern about transfusing adult platelets into a delicately balanced neonatal hemostatic system with relatively hypofunctional platelets as a potential etiology of this multifactorial outcome. In this retrospective review, we aimed to review transfusion practices and outcomes, including thrombosis, in the neonatal intensive care unit (NICU) at the University of Arizona, Tucson in comparison to the PlaNeT2 study. Methods After Institutional Review Board approval, we reviewed the electronic medical records of all neonates born at < 32 weeks gestation in the NICU or transferred to the NICU within 48 hours of birth between January 1, 2018 and December 31, 2019, corresponding to one year pre- and one year post-publication of the PlaNet2 study. For each patient record reviewed, we collected demographics, reviewed whether transfusions occurred, and documented pre-transfusion hemoglobin, platelet count, international normalized ratio, fibrinogen activity level, and clinical outcomes. We recorded the number of transfusions of each product, clinical central lines placed, and imaging studies performed to detect thrombosis. These data were analyzed using descriptive analysis. Results There were 127 neonates born During the two-year period of this review, 60% of patients had at least one pRBC transfusion, 13% of patients had at least one platelet transfusion, and 6% of patients had at least one fresh frozen plasma (FFP) transfusion. A total of 35 platelet, 370 pRBC, and 11 FFP transfusions were administered with a mean total of 3.3 total transfusions per patient. The mean and median pre-transfusion platelet counts were 79,300/uL and 52,000/uL, respectively. The mean and median pre-transfusion hemoglobin values were 12.1 g/dL and 12.2 g/dL, respectively. Only two of 35 platelet transfusions were given after the publication of the PlaNeT2 study in January 2019; however, both transfusions were given for neonates with pre-transfusion platelet counts >50,000/uL. There were no arterial or venous thrombosis events noted in our population even though 108 patients (85%) had a peripherally inserted central catheters for an average of 12 days, 32 (25%) had umbilical arterial catheters for an average of 7 days, and 66 (52%) had umbilical vein catheters placed for an average of 6 days. Conclusions The majority of transfusions given to neonates born at Disclosures No relevant conflicts of interest to declare.

Background and objective Pulmonary Hypertension (pH) is a chronic progressive disease. Endothelial cells (EC) play a central and critical role in the initiation and progression of pH. The NF-κB family (NF-κB1 (p50/p105), NF-κB2 (p52/p100), RelA (p65), RelB, and C-Rel) regulates a wide array of genes involved in inflammatory responses, cell proliferation, and survival. The involvement of specific NF-κB family members in the pathogenesis of hypoxia-induced pH remains to be determined. The objective of this study was to assess the specific role of individual NF-κB family members in mediating endothelial cell responses to hypoxia and its downstream effect on smooth muscle cell proliferation. Methods and results NF-κB family members’ expression were selectively reduced by siRNA in human pulmonary microvascular endothelial cells. Cells were then exposed to hypoxia (1%) for 24 h. Endothelin1, ICAM1 gene expression and Stat1 and Stat3 phosphorylation were assessed. Smooth muscle cells (SMC) proliferation was assessed by culturing them with EC conditioned media. Reduction of either NF-κB2 or RelA in EC, led to a significant decrease in Endothelin1 and ICAM1 gene expression. C-Rel knockdown resulted in a significant increase in phosphorylated STAT1; both C-Rel and RelA knockdown significantly decreased phosphorylated STAT3 in EC. There was a significant reduction in SMC proliferation, and AKT/ERK phosphorylation in SMC, when cultured in RelA knockdown, EC conditioned media. Conclusion RelA in EC plays crucial role in hypoxia induced vascular remodeling and development of pH. Targeting RelA in EC alleviates SMC proliferation as well as inflammation related processes.

Periventricular leukomalacia (PVL), a brain injury affecting premature infants is commonly associated with cerebral palsy. PVL results from hypoxia-ischemia (HI) with or without infection and is characterized by white matter necrotic lesions, hypomyelination, microglial activation, astrogliosis, and neuronal death. It is important to study a PVL mouse model that mimics human PVL in symptomatology, anatomic and molecular basis. In our neonate mice model, bilateral carotid arteries were temporary ligated at P5 followed by hypoxic exposure (FiO2 of 8% for 20 min.). At P5 in mice, the white matter is more vulnerable to HI injury than the grey matter. In our PVL model, mice suffer from significant hind limb paresis, incoordination and feeding difficulties. Histologically they present with ventriculomegally, white matter loss, microglial activation and neuronal apoptosis. HI injury increases proinflammtory cytokines, activates NF-kB, activates microglia and causes nitrative stress. All these inflammatory mediators lead to oligodendroglial injury and white matter loss. Neurobehavioral analysis in the PVL mice model at P60 showed that the HI group had a significant decrease in hind limb strength, worsening rotarod testing and worsening performance in the open field test. This new PVL model has great advantages far beyond just mimicking human PVL in clinical features and histopathology. Long term survival, the development of cerebral palsy and the ability of using this model in transgenic animals will increase our understanding of the mechanistic pathways underlying PVL and defining specific targets for the development of suitable therapeutics.

Point-of-care ultrasound (POC-US) is increasingly used especially in emergency and critical-care medicine. It is focused, quick and does not expose patients to ionizing radiation. It encompasses all organ systems and has well-defined indications. Lung ultrasound (LUS) represents one of the most exciting applications in the field of POC-US. It is particularly important to emphasize the role of LUS in neonatology due to the specific pathology inherent in lung immaturity as well as in the particular sensitivity of neonates to repeated radiation exposure. One of the main barriers to the more extensive use of the ultrasound technology is a lack of efficient and attractive training solutions followed by the structured quality-check assurance. In an effort to help bridge this gap, based on the most current literature, we developed creative and intuitive neonatal LUS algorithms. We hope they can serve as a clinical imaging guidelines and a valuable complement to the history and physical exam.

Background. Caffeine is widely used to treat apnea of prematurity. Here, we evaluated the efficacy of early caffeine (1-2 DOL) in decreasing the incidence of adverse neonatal outcomes.Methods. A retrospective cohort was used to compare the neonatal morbidity of 150 preterm neonates with gestational age ≤29 weeks. Infants were divided into 3 groups based on the initiation timing of caffeine therapy; (1) early caffeine (1-2 DOL), (2) late caffeine (3–7 DOL), and (3) very late caffeine (≥8 DOL).Results. The neonatal outcomes of early caffeine were comparable with those of the late caffeine group. Moreover, when comparing the neonatal morbidity of the very late caffeine group with that of the early caffeine group, multivariable logistic regression analyses were performed. We found that the timing of caffeine did not influence the risk of BPD (OR, 0.393; CI, 0.126–1.223;p=0.107), but birthweight did (OR, 0.996; CI, 0.993–0.999;p=0.018) in these infants.Conclusion. Neonatal outcomes of preterm infants were comparable whether caffeine was administered early or late in the first 7 DOL. The risk of BPD in infants receiving caffeine after 8 DOL was irrespective of delayed treatment with caffeine. Our results clearly demonstrate the need for further studies before caffeine prophylaxis can be universally recommended.

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P< 0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P < 0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.

Hypoxia leads to free radical production, which has a pivotal role in the pathophysiology of pulmonary hypertension (PH). We hypothesized that treatment with extracellular superoxide dismutase (EC-SOD) could ameliorate the development of PH induced by hypoxia. In vitro studies using pulmonary microvascular endothelial cells showed that cells transfected with EC-SOD had significantly less accumulation of xanthine oxidase and reactive oxygen species than nontransfected cells after hypoxia exposure for 24 h. To study the prophylactic role of EC-SOD, adult male wild-type (WT) and transgenic (TG) mice, with lung-specific overexpression of human EC-SOD (hEC-SOD), were exposed to fraction of inspired oxygen (FiO(2)) 10% for 10 d. After exposure, right ventricular systolic pressure (RVSP), right ventricular mass (RV/S + LV), pulmonary vascular wall thickness (PVWT) and pulmonary artery contraction/relaxation were assessed. TG mice were protected against PH compared with WT mice with significantly lower RVSP (23.9 ± 1.24 versus 47.2 ± 3.4), RV/S + LV (0.287 ± 0.015 versus 0.335 ± 0.022) and vascular remodeling, indicated by PVWT (14.324 ± 1.107 versus 18.885 ± 1.529). Functional studies using pulmonary arteries isolated from mice indicated that EC-SOD prevents hypoxia-mediated attenuation of nitric oxide-induced relaxation. Therapeutic potential was assessed by exposing WT mice to FiO(2) 10% for 10 d. Half of the group was transfected with plasmid containing cDNA encoding human EC-SOD. The remaining animals were transfected with empty vector. Both groups were exposed to FiO(2) 10% for a further 10 d. Transfected mice had significantly reduced RVSP (18.97 ± 1.12 versus 41.3 ± 1.5), RV/S + LV (0.293 ± 0.012 versus 0.372 ± 0.014) and PVWT (12.51 ± 0.72 versus 18.98 ± 1.24). On the basis of these findings, we concluded that overexpression of EC-SOD prevents the development of PH and ameliorates established PH.

Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of 18FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.