Neonatal Necrotizing Enterocolitis: An Update on Pathophysiology, Treatment, and Prevention.

Necrotizing enterocolitis (NEC) is a life-threatening disease predominantly affecting premature and very low birth weight infants resulting in inflammation and necrosis of the small bowel and colon and potentially leading to sepsis, peritonitis, perforation, and death. Numerous research efforts have been made to better understand, treat, and prevent NEC. This review explores a variety of factors involved in the pathogenesis of NEC (prematurity, low birth weight, lack of human breast milk exposure, alterations to the microbiota, maternal and environmental factors, and intestinal ischemia) and reports treatment modalities surrounding NEC, including pain medications and common antibiotic combinations, the rationale for these combinations, and recent antibiotic stewardship approaches surrounding NEC treatment. This review also highlights the effect of early antibiotic exposure, infections, proton pump inhibitors (PPIs), and H2 receptor antagonists on the microbiota and how these risk factors can increase the chances of NEC. Finally, modern prevention strategies including the use of human breast milk and standardized feeding regimens are discussed, as well as promising new preventative and treatment options for NEC including probiotics and stem cell therapy.

Hypertension induced by pregnancy and neonatal outcome: Results from a retrospective cohort study in preterm under 34 weeks.

OBJECTIVE: The present study seeks to assess the impact of gestational hypertensive disorders on premature newborns below 34 weeks and to establish the main morbidities and mortality in the neonatal period and at 18 months. MATERIALS AND METHODS: A retrospective observational study was carried out with 695 premature newborns of gestational age (GA) between 24 and 33 weeks and 6 days, born alive in the Neonatal ICU of Brasília's Mother and Child Hospital (HMIB), in the period from January 1, 2014, to July 31, 2019. In total, 308 infants were born to hypertensive mothers (G1) and 387 to normotensive mothers (G2). Twin pregnancies and diabetic patients with severe malformations were excluded. Outcomes during hospitalization and outcomes of interest were evaluated: respiratory distress syndrome (RDS), brain ultrasonography, diagnosis of bronchopulmonary dysplasia (BPD), diagnosis of necrotizing enterocolitis, retinopathy of prematurity, breastfeeding rate at discharge, survival at discharge and at 18 months of chronological age and relationship between weight and gestational age. RESULTS: Newborns with hypertensive mothers had significantly lower measurements of birth weight and head circumference. The G1 group had a higher risk small for gestational age (OR 2.4; CI 95% 1.6-3.6; p <0.00), as well as a greater risk of being born with a weight less than 850 g (OR 2.4; 95% CI 1.2-3.5; p <0.00). Newborns of mothers with hypertension presented more necrotizing enterocolitis (OR 2.0; CI 95% 1.1-3.7); however, resuscitation in the delivery room and the need to use surfactant did not differ between groups, nor did the length of stay on mechanical ventilation, or dependence on oxygen at 36 weeks of gestational age. Survival was better in newborns of normotensive mothers, and this was a protective factor against death (OR 0.7; 95% CI 0.5-0.9; p <0.01). In the follow-up clinic, survival at 18 months of chronological age was similar between groups, with rates of 95.3% and 92.1% among hypertensive and normotensive mothers, respectively. Exclusive breastfeeding at discharge was 73.4% in the group of hypertensive women and 77.3% in the group of normotensive mothers. There were no significant differences between groups. CONCLUSION: Among the analyzed outcomes, arterial hypertension during pregnancy can increase the risk of low weight, small babies for gestational age (SGA), deaths in the neonatal period and enterocolitis, with no differences in weight and survival at 18 months of chronological age. Arterial hypertension presents a high risk of prematurity in the neonatal period, with no difference at 18 months of age.

Necrotizing Enterocolitis: Overview on In Vitro Models.

Necrotizing enterocolitis (NEC) is a gut inflammatory disorder which constitutes one of the leading causes of morbidity and mortality for preterm infants. The pathophysiology of NEC is yet to be fully understood; several observational studies have led to the identification of multiple factors involved in the pathophysiology of the disease, including gut immaturity and dysbiosis of the intestinal microbiome. Given the complex interactions between microbiota, enterocytes, and immune cells, and the limited access to fetal human tissues for experimental studies, animal models have long been essential to describe NEC mechanisms. However, at present there is no animal model perfectly mimicking human NEC; furthermore, the disease mechanisms appear too complex to be studied in single-cell cultures. Thus, researchers have developed new approaches in which intestinal epithelial cells are exposed to a combination of environmental and microbial factors which can potentially trigger NEC. In addition, organoids have gained increasing attention as promising models for studying NEC development. Currently, several in vitro models have been proposed and have contributed to describe the disease in deeper detail. In this paper, we will provide an updated review of available in vitro models of NEC and an overview of current knowledge regarding its molecular underpinnings.

Stress and corticotropin releasing factor (CRF) promote necrotizing enterocolitis in a formula-fed neonatal rat model.

The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p<0.001). Astressin prevented development of NEC in FF-stressed neonates (7.7% vs. 40.0%; p = 0.003). CRF and CRFR immunoreactivity increased in the ileum of neonates with NEC compared to dam-fed controls or FF unstressed pups. Immunoblotting confirmed increased TLR4 protein levels in FF stressed (NEC model) animals vs. controls, and Astressin treatment restored TLR4 to control levels. Peripheral CRF may serve as specific pharmacologic target for the prevention and treatment of NEC.

Doppler ultrasound assessment of splanchnic perfusion and heart rate for the detection of necrotizing enterocolitis.

PURPOSE: Monitoring disease progression is crucial to improve the outcome of necrotizing enterocolitis (NEC). A previous study indicates that intestinal wall flow velocity was reduced in NEC pups from the initial stages of the disease. This study aims to investigate whether splanchnic perfusion via the superior mesenteric artery (SMA) (i) is altered during NEC development and (ii) can be used as a monitoring tool to assess disease progression. METHODS: NEC was induced in C57BL/6 mice via gavage feeding of formula, hypoxia, and oral lipopolysaccharide, from postnatal day 5 (P5) to P9 (AUP: 32,238). Breastfed littermates served as controls. Doppler ultrasound (U/S) of bowel loops was performed daily. Intestinal wall perfusion was calculated as average flow velocity (mm/s) of multiple abdominal regions. Groups were compared using one-way ANOVA. RESULTS: The SMA flow velocity was not altered during the initial stage of NEC development, but become significantly reduced at P8 when the intestinal disease was more advanced. These changes occurred concomitantly with an increase in heart rate. CONCLUSIONS: NEC is associated with intestinal hypo-perfusion at the periphery and flow in the SMA is reduced during the later stages of disease indicating the presence of intestinal epithelium damage. This study contributes to understanding NEC pathophysiology and illustrates the value of Doppler U/S in monitoring disease progression.

Necrotizing enterocolitis intestinal barrier function protection by antenatal dexamethasone and surfactant-D in a rat model.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common gastrointestinal disorder in premature neonates. Possible therapeutic approaches are centered on promoting maturation of the gastrointestinal mucosal barrier. Studies have demonstrated that antenatal administration of corticosteroids can decrease NEC incidence and mortality. METHODS: Pregnant rat dams were administered dexamethasone 48 h prior to delivery. The pups were subjected to an experimental NEC-like injury protocol. Ileal tissues and sera were collected and evaluated for inflammatory cytokines, gut permeability and expressions and localizations of tight junction proteins, and surfactant protein-D by immunohistochemistry/immunofluorescent staining. Intestinal epithelial cells (IEC-6) were pretreated with SP-D to examine the effect of SP-D on tight junction protein expressions when challenged with platelet-activating factor and lipopolysaccharide to model proinflammatory insults. RESULTS: Antenatal dexamethasone reduced systemic inflammation, preserved intestinal barrier integrity, and stimulated SP-D expression on the intestinal mucosal surface in pups exposed to NEC-like injury. Pretreatment of SP-D blocked platelet-activating factor/lipopolysaccharide-induced tight junction disruption in IEC-6 cells in vitro. CONCLUSIONS: Antenatal dexamethasone preserves the development of intestinal mucosal barrier integrity and reduces incidence and morbidity from an experimental NEC-like injury model. Dexamethasone upregulation of intestinal SP-D-protective effects on tight junction proteins. IMPACT: Antenatal administration of dexamethasone can function in concert with intestinal surfactant protein-D to decrease systemic inflammatory responses, and protect intestinal barrier integrity in a neonatal rat model of NEC. A novel role of intestinal SP-D in preserving tight junction protein structures under inflammatory conditions. We describe the intestinal SP-D-an overlooked role of antenatal dexamethasone in neonatal NEC?

Utility of volatile organic compounds as a diagnostic tool in preterm infants.

BACKGROUND: Volatile organic compounds (VOCs) are hydrocarbons that originate within different healthy and diseased tissues. VOCs can be secreted into the circulation and then excreted in the urine and faeces. In the lungs, VOCs are locally produced and can be detected in exhaled breath. VOCs can be identified using non-invasive techniques, which make their use in preterm infants safe and desirable. METHODS: A systematic search of the literature in PubMed, Embase and Web of Science was conducted looking for VOCs techniques and diagnostic performance in preterm infants. A total of 50 articles identified with only seven papers were included in the final analysis in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). RESULTS: VOCs could diagnose necrotising enterocolitis up to 4 days before a clinical diagnosis; for late onset sepsis, up to 3 days before; and for bronchopulmonary dysplasia, up to 2 weeks before. In addition to these diagnostic uses, VOCs analysis could also distinguish breastfed from formula-fed preterm neonates in the first month of life. CONCLUSION: VOCs analysis is a non-invasive tool that makes the use in preterm infants of preference. VOCs analytic techniques require more research and consensus between researchers to overcome their limitations. IMPACT: Volatile organic compounds are hydrocarbons that can separate between healthy and diseased states in preterm infants. Biomarker panels developed from volatile organic compounds are potential diagnostic tools. The non-invasive nature of acquiring volatile organic compounds markers make it desirable in the paediatric patients. Research into exact chemical components of the volatile organic compounds can inform about the pathophysiology of disease in preterm infants. More robust longitudinal studies with repeated experiments are required before volatile organic compounds can be applied in clinical practice.

Low descending aorta flow is associated with adverse feeding outcomes in neonates with small left-sided structures.

Feeding intolerance and necrotizing enterocolitis (NEC) cause significant morbidity in neonates with duct-dependent systemic circulations. Whether these complications are associated with low blood flow to the bowel is unproven. The aim of this study was to determine whether low descending aortic (DAO) flow is associated with adverse feeding outcomes in neonates with small left-sided structures, including borderline left ventricle and hypoplastic left heart syndrome (HLHS). The cardiac magnetic resonance (CMR) imaging studies and abdominal Doppler ultrasound profiles prior to any cardiac interventions in neonates with small left-sided structures were analyzed. Descending aortic flows, indexed to body surface area, were collected. Medical charts were reviewed for a composite outcome of feeding intolerance and/or NEC. Among the 51 enrolled study patients (mean age 4.6, SD 4.5 days), 13 experienced the composite outcome (feeding intolerance in 13, NEC in 2). The mean DAO flow in patients who experienced the composite outcome was 0.89 L/min/m2 (SD 0.33 L/min/m2), compared to 1.23 L/min/m2 (SD 0.41 L/min/m2) in those that did not (p = 0.007). A DAO flow of 0.91 L/min/m2 identified patients who experienced feeding intolerance or NEC with a sensitivity of 61% and a specificity of 76%. Doppler ultrasound metrics of DAO flow did not correlate with DAO flow or predict adverse feeding outcomes. Low DAO flow is associated with adverse outcomes, including feeding intolerance and NEC, in neonates with small left-sided structures. Heightened clinical vigilance towards feeding complications in patients with low DAO flow is recommended.

Gut transit time, using radiological contrast imaging, to predict early signs of necrotizing enterocolitis.

BACKGROUND: Immature gut motility in preterm neonates may be a risk factor for necrotizing enterocolitis (NEC). Using preterm pigs as a model for infants, we hypothesized that intestinal dysmotility precedes NEC development. METHODS: Eighty-five preterm pigs were fed increasing amounts of milk diets to induce NEC lesions, as detected at autopsy on day 5. Gut transit time was determined on day 4 by x-ray imaging after oral intake of contrast solution. RESULTS: No clinical or radiological signs of NEC were detected on day 4, but macroscopic NEC lesions were recorded in 59% of pigs (n = 50) on day 5. Relative to pigs without NEC (noNEC, n = 35), pigs with small intestinal lesions (siNEC, n = 18) showed delayed stomach emptying time (StEmpty) and time for contrast to reach cecum (ToCecum) already on day 4. Pigs with lesions only in colon (coNEC, n = 20) showed more diarrhea, shorter ToCecum time, but longer small intestinal emptying time (SiEmpty). ToCecum time predicted siNEC and coNEC lesions with a receiver-operator characteristic area under the curve of 78-81%. CONCLUSIONS: Region-dependent changes in gut transit time is associated with early NEC development in preterm pigs. How gut dysmotility is related to NEC in preterm infants requires further investigations. IMPACT: Using preterm pigs as a model for preterm infants, we show that gut transit time, using serial x-ray contrast imaging, was changed in individuals with NEC-like lesions before they showed the typical radiological signs of NEC. Thus prolonged transit time across the entire gut was recorded when NEC lesions appeared in the small intestine but not when lesions were detected only in the colon. Until now, recordings of food transit have mainly investigated changes in the upper gut. Using serial x-rays, this study describes food transit across the entire gut and documents a region-dependent effect of NEC lesions on gut transit changes in preterm individuals. The findings provide proof of concept for use of x-ray contrast imaging as a tool to monitor gut transit in preterm pigs as models for infants. Delayed passage across the entire gut may be an early sign of small intestinal NEC, at least in pigs. More studies are needed to confirm relations in infants. In the future, it might be possible to use x-ray contrast imaging in preterm infants to better understand gut motility in relation to early NEC progression and need for medical NEC treatment.

Necrotizing Enterocolitis-like Pneumatosis Intestinalis in an Infant With COVID-19.

We report an infant with COVID-19 who presented with bloody stools, lethargy and imaging findings significant for pneumatosis intestinalis. The infant was treated with conservative therapy, including resuscitation, bowel rest and intravenous antibiotics, successfully avoiding surgical intervention.

The Role of Human Milk in Decreasing Necrotizing Enterocolitis Through Modulation of the Infant Gut Microbiome: A Scoping Review.

BACKGROUND: Necrotizing enterocolitis is associated with a high incidence of morbidity and mortality in premature infants. Human milk minimizes necrotizing enterocolitis risk, although the mechanism of protection is not thoroughly understood. Increasingly, dysbiosis of the infant gut microbiome, which is affected by infant diet, is hypothesized to play a role in necrotizing enterocolitis pathophysiology. RESEARCH AIM: The aim of this scoping review was to summarize the state of the science regarding the hypothesis that the gut microbiome composition is a mediator of the relationship between human milk and decreased incidence of necrotizing enterocolitis within a sample of human infants. METHODS: Electronic databases and reference lists were searched for peer-reviewed primary research articles addressing the link between human milk, gut microbiome composition, and subsequent incidence of necrotizing enterocolitis among human infants. RESULTS: A total of four studies met criteria for inclusion in this review. Of these, evidence supporting the link between human milk, gut microbiome composition, and necrotizing enterocolitis was found in two (50%) studies. CONCLUSION: Some evidence linking all three variables is provided in this review. Given the small number of available studies, and the limitations of those studies, more research is urgently needed to thoroughly understand the protection against necrotizing enterocolitis gained through the provision of human milk.

Low cardiac output measured by bioreactance and adverse outcome in preterm infants with birth weight less than 1250 g.

BACKGROUND: Recently a new continuous non-invasive cardiac output measurement, bioreactance, has become available. Bioreactance measurement of cardiac output has been shown to correlate with left ventricular output detected by echocardiography in healthy term and preterm neonates. AIMS: Our aim was to correlate cardiac output measurements by bioreactance in the first 48 h of life with adverse outcomes attributable to hypoperfusion (peri/intraventricular haemorrhage (PIVH) and/or necrotising enterocolitis (NEC)) in the cohort of extremely preterm infants. STUDY DESIGN: A prospective observational cohort study. SUBJECTS: Preterm infants with birth weight less than 1250 g. OUTCOME MEASURES: Cardiac output was measured between six and 48 h of age by bioreactance. Our primary outcome was a difference in cardiac output between infants with an adverse outcome attributable to hypoperfusion (Group 1), and infants without the predefined adverse outcome (Group 2). RESULTS: There were 39 infants enrolled in the study. There were six infants in Group 1. These infants had a significantly lower minimal cardiac output measurement compared to Group 2 (mean 36.7 ml/kg/min vs 64.5 ml/kg/min, p = .0006). The mean cardiac output in Group 1 was significantly lower on day one of life, followed by a significant increase in cardiac output on day two of life compared to Group 2. CONCLUSIONS: Infants with birth weight less than 1250 g and PIVH and/or NEC had significantly lower cardiac output compared to infants without these complications on day one of life. This low cardiac output was then followed by a significant increase on day two of life.

Intervention and Outcome for Neonatal Hypotension.

Many observational studies have shown that infants with blood pressures (BPs) that are in the lower range for their gestational age tend to have increased complications such as an increased rate of significant intraventricular hemorrhage and adverse long-term outcome. This relationship does not prove causation nor should it create an indication for treatment. However, many continue to intervene with medication for low BP on the assumption that an increase in BP will result in improved outcome. Only adequately powered prospective randomized controlled trials can answer the question of whether individual treatments of low BP are beneficial.

Probiotics Reduce Mortality and Morbidity in Preterm, Low-Birth-Weight Infants: A Systematic Review and Network Meta-analysis of Randomized Trials.

BACKGROUND & AIMS: We aimed to compare the effectiveness of single- vs multiple-strain probiotics in a network meta-analysis of randomized trials. METHODS: We searched MEDLINE, Embase, Science Citation Index Expanded, CINAHL, Scopus, Cochrane CENTRAL, BIOSIS Previews, and Google Scholar through January 1, 2019, for studies of single-strain and multistrain probiotic formulations on the outcomes of preterm, low-birth-weight neonates. We used a frequentist approach for network meta-analysis and the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to assess the certainty of evidence. Primary outcomes included all-cause mortality, severe necrotizing enterocolitis (NEC) (Bell stage II or more), and culture-proven sepsis. RESULTS: We analyzed data from 63 trials involving 15,712 preterm infants. Compared with placebo, a combination of 1 or more Lactobacillus species (spp) and 1 or more Bifidobacterium spp was the only intervention with moderate- or high-quality evidence of reduced all-cause mortality (odds ratio [OR], 0.56; 95% confidence interval [CI], 0.39-0.80). Among interventions with moderate- or high-quality evidence for efficacy compared with placebo, combinations of 1 or more Lactobacillus spp and 1 or more Bifidobacterium spp, Bifidobacterium animalis subspecies lactis, Lactobacillus reuteri, or Lactobacillus rhamnosus significantly reduced severe NEC (OR, 0.35 [95% CI, 0.20-0.59]; OR, 0.31 [95% CI, 0.13-0.74]; OR, 0.55 [95% CI, 0.34-0.91]; and OR, 0.44 [95% CI, 0.21-0.90], respectively). There was moderate- or high-quality evidence that combinations of 1 or more Lactobacillus spp and 1 or more Bifidobacterium spp and Saccharomyces boulardii reduced the number of days to reach full feeding (mean reduction of 3.30 days [95% CI, reduction of 5.91-0.69 days]). There was moderate- or high-quality evidence that, compared with placebo, the single-species product B animalis subsp lactis or L reuteri significantly reduced duration of hospitalization (mean reduction of 13.00 days [95% CI, reduction of 22.71-3.29 days] and mean reduction of 7.89 days [95% CI, reduction of 11.60-4.17 days], respectively). CONCLUSIONS: In a systematic review and network meta-analysis of studies to determine the effects of single-strain and multistrain probiotic formulations on outcomes of preterm, low-birth-weight neonates, we found moderate to high evidence for the superiority of combinations of 1 or more Lactobacillus spp and 1 or more Bifidobacterium spp vs single- and other multiple-strain probiotic treatments. The combinations of Bacillus spp and Enterococcus spp, and 1 or more Bifidobacterium spp and Streptococcus salivarius subsp thermophilus, might produce the largest reduction in NEC development. Further trials are needed.

The Paneth Cell: The Curator and Defender of the Immature Small Intestine.

Paneth cells were first described in the late 19th century by Gustav Schwalbe and Josef Paneth as columnar epithelial cells possessing prominent eosinophilic granules in their cytoplasm. Decades later there is continued interest in Paneth cells as they play an integral role in maintaining intestinal homeostasis and modulating the physiology of the small intestine and its associated microbial flora. Paneth cells are highly specialized secretory epithelial cells located in the small intestinal crypts of Lieberkühn. The dense granules produced by Paneth cells contain an abundance of antimicrobial peptides and immunomodulating proteins that function to regulate the composition of the intestinal flora. This in turn plays a significant role in secondary regulation of the host microvasculature, the normal injury and repair mechanisms of the intestinal epithelial layer, and the levels of intestinal inflammation. These critical functions may have even more importance in the immature intestine of premature infants. While Paneth cells begin to develop in the middle of human gestation, they do not become immune competent or reach their adult density until closer to term gestation. This leaves preterm infants deficient in normal Paneth cell biology during the greatest window of susceptibility to develop intestinal pathology such as necrotizing enterocolitis (NEC). As 10% of infants worldwide are currently born prematurely, there is a significant population of infants contending with an inadequate cohort of Paneth cells. Infants who have developed NEC have decreased Paneth cell numbers compared to age-matched controls, and ablation of murine Paneth cells results in a NEC-like phenotype suggesting again that Paneth cell function is critical to homeostasis to the immature intestine. This review will provide an up to date and comprehensive look at Paneth cell ontogeny, the impact Paneth cells have on the host-microbial axis in the immature intestine, and the repercussions of Paneth cell dysfunction or loss on injury and repair mechanisms in the immature gut.

A randomized controlled trial protocol comparing the feeds of fresh versus frozen mother's own milk for preterm infants in the NICU.

BACKGROUND: Necrotizing enterocolitis (NEC) is the leading cause of death among preterm infants born at < 30 weeks' gestation. The incidence of NEC is reduced when infants are fed human milk. However, in many neonatal intensive care units (NICUs), it is standard practice to freeze and/or pasteurize human milk, which deactivates bioactive components that may offer additional protective benefits. Indeed, our pilot study showed that one feed of fresh mother's own milk per day was safe, feasible, and can reduce morbidity in preterm infants. To further evaluate the benefits of fresh human milk in the NICU, a randomized controlled trial is needed. METHODS: Our prospective multicenter, double-blinded, randomized, controlled trial will include infants born at < 30 weeks' gestation and admitted to one of 29 tertiary NICUs in China. Infants in the intervention (fresh human milk) group (n = 1549) will receive at least two feeds of fresh human milk (i.e., within 4 h of expression) per day from the time of enrollment until 32 weeks' corrected age or discharge to home. Infants in the control group (n = 1549) will receive previously frozen human milk following the current standard protocols. Following informed consent, enrolled infants will be randomly allocated to the control or fresh human milk groups. The primary outcome is the composite outcome mortality or NEC ≥ stage 2 at 32 weeks' corrected age, and the secondary outcomes are mortality, NEC ≥ stage 2, NEC needing surgery, late-onset sepsis, retinopathy of prematurity (ROP), bronchopulmonary dysplasia (BPD), weight gain, change in weight, increase in length, increase in head circumference, time to full enteral feeds, and finally, the number and type of critical incident reports, including feeding errors. DISCUSSION: Our double-blinded, randomized, controlled trial aims to examine whether fresh human milk can improve infant outcomes. The results of this study will impact both Chinese and international medical practice and feeding policy for preterm infants. In addition, data from our study will inform changes in health policy in NICUs across China, such that mothers are encouraged to enter the NICU and express fresh milk for their infants. TRIAL REGISTRATION: Chinese Clinical Trial Registry; #ChiCTR1900020577; registered January 1, 2019; http://www.chictr.org.cn/showprojen.aspx?proj=34276.

Lactoferrin Reduces Necrotizing Enterocolitis Severity by Upregulating Intestinal Epithelial Proliferation.

INTRODUCTION: Necrotizing enterocolitis (NEC) is a devastating intestinal illness in premature infants characterized by severe intestinal inflammation. Despite medical interventions, NEC mortality remains alarmingly high, which necessitates improved therapies. Lactoferrin is among the most abundant proteins in human milk and has important immunomodulatory functions. While previous studies have indicated protective effects of lactoferrin against neonatal sepsis and NEC, the underlying mechanism remains unclear. We hypothesize that lactoferrin downregulates inflammation and upregulates proliferation in intestinal epithelium during NEC injury. MATERIALS AND METHODS: NEC was induced by hypoxia, gavage feeding of hyperosmolar formula and lipopolysaccharide between postnatal day P5 and P9 (n = 8). Breastfed mice were used as control (n = 7). Lactoferrin (0.3 g/kg/day) was administered once daily by gavage from P6 to P8 in both NEC (NEC + Lac; n = 9) and control mice (Cont + Lac; n = 5). Distal ileum was harvested on P9 and analyzed for disease severity, inflammation, and proliferation. Groups were compared using one-way ANOVA and t-test appropriately; p < 0.05 was considered significant. RESULTS: Compared to NEC group, lactoferrin-treated NEC mice had reduced disease severity, reduced inflammation markers IL-6 and TNF-α expression and increased intestinal stem cell marker Lgr5 + expression. Lactoferrin-treated NEC mice exhibited increased nuclear ß-catenin, indicating upregulated Wnt pathway, and increased Ki67 positivity, suggesting enhanced proliferation. Furthermore, lactoferrin administration to control mice did not affect intestinal inflammation as well as Lgr5 + stem cell expression and epithelial proliferation. This supports the safety of lactoferrin administration and indicates that the beneficial effects of lactoferrin are present when intestinal injury such as NEC is present. CONCLUSION: Lactoferrin administration reduces the intestinal injury in experimental NEC by downregulating inflammation and upregulating cell proliferation. This beneficial effect of lactoferrin in stimulating cell proliferation is mediated by the Wnt pathway. This experimental study provides insights on the mechanism of action of lactoferrin in NEC and the role of lactoferrin in enteral feeding.

Liver Organoids Generated from Mice with Necrotizing Enterocolitis Have Reduced Regenerative Capacity.

INTRODUCTION: Necrotizing enterocolitis (NEC) is one of the most severe gastrointestinal diseases in infancy. NEC can cause metabolic derangements, multi-organ injury including severe liver damage. The mechanism leading to hepatic damage in NEC remains unclear. The aim of this study is to establish and characterize liver organoids from NEC mice. MATERIALS AND METHODS: Following ethical approval (#44032), we induced experimental NEC from postnatal day 5 (P5) to P9 using C57BL/6 mice pups. NEC was induced by gavage formula feeding, gavage lipopolysaccharide (LPS) administration, and hypoxia. Breastfed pups were used as control. On P9, NEC and control pups were sacrificed and liver tissue was harvested and organoids were generated. Organoid size was recorded daily (day 2-4) by measuring their surface area and organoid growth was calculated. RNA was extracted on day 4 after liver organoid generation. RESULTS: Organoid growth rate was significantly lower in NEC liver organoids compared to control liver organoids. mRNA expression of liver progenitor cells markers of LGR5 and SOX9 was lower in NEC liver organoids compared to control liver organoids. Similarly, expression of proliferation markers of Ki67 and PCNA was lower in NEC liver organoids. CONCLUSION: We report a novel technique to generate liver organoids during NEC. These organoids are characterized by reduced progenitor cells, reduced proliferation, and overall impaired regenerative capacity. Liver progenitor cells are important targets to prevent liver damage in NEC and promote recovery.

Predicting intestinal recovery after necrotizing enterocolitis in preterm infants.

BACKGROUND: Intestinal recovery after NEC is difficult to predict in individuals. We evaluated whether several biomarkers predict intestinal recovery after NEC in preterm infants. METHODS: We measured intestinal tissue oxygen saturation (rintSO2) and collected urinary intestinal-fatty acid binding protein (I-FABPu) levels 0-24 h and 24-48 h after NEC onset, and before and after the first re-feed. We assessed intestinal recovery in two ways: time to full enteral feeding (FEFt; below or equal/above group's median) and development of post-NEC complications (recurrent NEC/post-NEC stricture). We determined whether the rintSO2, its range, and I-FABPu differed between groups. RESULTS: We included 27 preterm infants who survived NEC (Bell's stage ≥ 2). Median FEFt was 14 [IQR: 12-23] days. Biomarkers only predicted intestinal recovery after the first re-feed. Mean rintSO2 ≥ 53% combined with mean rintSO2range ≥ 50% predicted FEFt < 14 days with OR 16.7 (CI: 2.3-122.2). The rintSO2range was smaller (33% vs. 51%, p < 0.01) and I-FABPu was higher (92.4 vs. 25.5 ng/mL, p = 0.03) in case of post-NEC stricture, but not different in case of recurrent NEC, compared with infants without complications. CONCLUSION: The rintSO2, its range, and I-FABPu after the first re-feed after NEC predicted intestinal recovery. These biomarkers have potential value in individualizing feeding regimens after NEC.