Hydrogen Sulfide Improves Outcomes in a Murine Model of Necrotizing Enterocolitis via the Cys440 Residue on Endothelial Nitric Oxide Synthase.

BACKGROUND: Hydrogen sulfide (H2S) has been shown to improve outcomes in a murine model of necrotizing enterocolitis (NEC). There is evidence in humans that H2S relies on endothelial nitric oxide synthase (eNOS) to exert its protective effects, potentially through the persulfidation of eNOS at the Cysteine 443 residue. We obtained a novel mouse strain with a mutation at this residue (eNOSC440G) and hypothesized that this locus would be critical for GYY4137 (an H2S donor) to exert its protective effects. METHODS: Necrotizing enterocolitis was induced in 5-day old wild type (WT) and eNOSC440G mice using intermittent exposure to hypoxia and hypothermia in addition to gavage formula feeds. On postnatal day 9, mice were humanely euthanized. Data collected included daily weights, clinical sickness scores, histologic lung injury, intestinal injury (macroscopically and histologically), and intestinal perfusion. During the NEC model, pups received daily intraperitoneal injections of either GYY4137 (50 mg/kg) or PBS (vehicle). Data were tested for normality and compared using t-test or Mann-Whitney, and a p-value <0.05 was considered significant. RESULTS: In WT mice, the administration of GYY4137 significantly improved clinical sickness scores, attenuated intestinal and lung injury, and improved mesenteric perfusion compared to vehicle (p < 0.05). In eNOSC440G mice, the treatment and vehicle groups had similar clinical sickness scores, intestinal and lung injury scores, and intestinal perfusion. CONCLUSIONS: GYY4137 administration improves clinical outcomes, attenuates intestinal and lung injury, and improves perfusion in a murine model of necrotizing enterocolitis. The beneficial effects of GYY4137 are dependent on the Cys440 residue of eNOS.

Age disparities in intestinal stem cell quantities: a possible explanation for preterm infant susceptibility to necrotizing enterocolitis.

PURPOSE: Preterm infants are more susceptible to necrotizing enterocolitis (NEC) than term Queryinfants. This may be due to a relative paucity of Lgr5+ or Bmi1+-expressing intestinal stem cells (ISCs) which are responsible for promoting intestinal recovery after injury. We hypothesized that the cellular markers of Lgr5+ and Bmi1+, which represent the two distinct ISC populations, would be lower in younger mice compared to older mice. In addition, we hypothesized that experimental NEC would result in a greater loss of Lgr5+ expression compared to Bmi1+ expression. METHODS: Transgenic mice with EGFP-labeled Lgr5 underwent euthanasia at 10 different time points from E15 to P56 (n = 8-11/group). Lgr5+-expressing ISCs were quantified by GFP ELISA and Bmi1+ was assessed by qPCR. In addition, Lgr5EGFP mice underwent experimental NEC via formula feeding and hypoxic and hypothermic stress. Additional portions of the intestine underwent immunostaining with anti-GFP or anti-Bmi1+ antibodies to confirm ELISA and PCR results. For statistical analysis, p < 0.05 was significant. RESULTS: Lgr5+ and Bmi1+expression was lowest in embryonal and early postnatal mice and increased with age in all segments of the intestine. Experimental NEC was associated with loss of Lgr5+-expressing ISCs but no significant change in Bmi1+ expression. CONCLUSION: Lgr5+ and Bmi1+ expression increase with age. Lgr5+-expressing ISCs are lower following experimental necrotizing enterocolitis while Bmi1+ expression remains relatively unchanged. Developing a targeted medical therapy to protect the low population of ISCs in preterm infants may promote tissue recovery and regeneration after injury from NEC.

A hydrogen-sulfide derivative of mesalamine reduces the severity of intestinal and lung injury in necrotizing enterocolitis through endothelial nitric oxide synthase.

Necrotizing enterocolitis (NEC) remains a devastating disease that affects preterm infants. Hydrogen sulfide (H2S) donors have been shown to reduce the severity of NEC, but the optimal compound has yet to be identified. We hypothesized that oral H2S-Mesalamine (ATB-429) would improve outcomes in experimental NEC, and its benefits would be dependent on endothelial nitric oxide synthase (eNOS) pathways. NEC was induced in 5-day-old wild-type (WT) and eNOS knockout (eNOSKO) pups by formula feeding and stress. Four groups were studied in both WT and eNOSKO mice: 1) breastfed controls, 2) NEC, 3) NEC + 50 mg/kg mesalamine, and 4) NEC + 130 mg/kg ATB-429. Mesalamine and ATB-429 doses were equimolar. Pups were monitored for sickness scores and perfusion to the gut was measured by Laser Doppler Imaging (LDI). After euthanasia of the pups, intestine and lung were hematoxylin and eosin-stained and scored for injury in a blind fashion. TLR4 expression was quantified by Western blot and IL-6 expression by ELISA. P < 0.05 was significant. Both WT and eNOSKO breastfed controls underwent normal development and demonstrated milder intestinal and pulmonary injury compared with NEC groups. For the WT groups, ATB-429 significantly improved weight gain, reduced clinical sickness score, and improved perfusion compared with the NEC group. In addition, WT ATB-429 pups had a significantly milder intestinal and pulmonary histologic injury when compared with NEC. ATB-429 attenuated the increase in TLR4 and IL-6 expression in the intestine. When the experiment was repeated in eNOSKO pups, ATB-429 offered no benefit in weight gain, sickness scores, perfusion, intestinal injury, pulmonary injury, or decreasing intestinal inflammatory markers. An H2S derivative of mesalamine improves outcomes in experimental NEC. Protective effects appear to be mediated through eNOS. Further research is warranted to explore whether ATB-429 may be an effective oral therapy to combat NEC.

Sildenafil attenuates intestinal injury in necrotizing enterocolitis independently of endothelial nitric oxide synthase.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease that impacts the intestine of premature infants. Sildenafil has shown benefit in colitis and ischemia/reperfusion models but has not been adequately studied in NEC. Sildenafil's best studied mechanism involves augmenting nitric oxide induced vasodilation. We hypothesized that sildenafil would improve outcomes during experimental NEC in an eNOS dependent manner. MATERIALS: NEC was induced in five-day old mouse pups with gavage formula feeds plus intermittent hypoxia and hypothermia. Using wild type (WT) mice, the route of sildenafil administration was studied in the following groups: (1) breastfed controls, (2) NEC + oral (PO) sildenafil, (3) NEC + PO vehicle, (4) NEC + intraperitoneal (IP) sildenafil, (5) NEC + IP vehicle. The eNOS KO groups studied included: (1) breastfed controls, (2) NEC + PO sildenafil, (3) NEC + PO vehicle. Data were tested for normality and compared using t-tests or Mann-Whitney with a p-value <0.05 considered significant. RESULTS: In WT mice, oral and IP sildenafil resulted in improved clinical outcomes compared to their respective vehicle group. Only orally administered sildenafil significantly improved perfusion to the intestine and protected it from macroscopic and histologic injury. When repeated in eNOS KO mice, oral sildenafil improved clinical scores and attenuated intestinal injury scores, despite no effect on intestinal perfusion. CONCLUSIONS: Sildenafil, when administered orally, improves clinical outcomes and protects the intestine in a murine model of experimental necrotizing enterocolitis. While sildenafil requires eNOS to impact mesenteric perfusion, it does not appear to be dependent on eNOS to attenuate intestinal injury.

Neurolysis for analgesia following pectus repair in a national cohort.

INTRODUCTION: Pectus excavatum and pectus carinatum are the most common chest wall deformities of childhood. Surgical repair can be complicated by post-operative analgesic challenges. Thoracic epidural analgesia, patient-controlled analgesia, and multimodal pain control are among the most common strategies. We sought to define the current utilization of intraoperative thoracic neurolysis, hypothesizing that this would minimize length of stay (LOS) and post-operative narcotic use with relatively higher proportion of non-narcotic post-operative analgesia. METHODS: We performed a retrospective review of the Pediatric Health Information System (PHIS) database between 2017 and 2020. We first identified patients who underwent a pectus repair via ICD-10-PCS codes. We used ICD-10-PCS codes 01580ZZ and 01584ZZ to identify those patients who underwent concomitant thoracic neurolysis. Statistical analyses were performed using R; p value less than 0.05 was considered significant. RESULTS: We identified 2979 patients who underwent a pectus repair. 184 underwent a concomitant thoracic nerve destruction procedure (6.7%); 13 were performed in 2017 (2.01%), 76 in 2018 (10.7%), and 84 in 2019 (9.6%). LOS was shorter in those patients who underwent neurolysis (mean=2.55 vs 3.73 days, SD=1.33 vs 1.78 days, p<0.001). There were fewer post-operative ICU admissions in neurolysis patients (3/184 vs. 193/2795, p = 0.003). The cost of procedures that included a neurolysis were higher, though not significantly so (mean=$24,885.64 vs $22,200.59). CONCLUSION: Thoracic neurolysis may be a useful analgesic strategy, expediating post-operative discharge and potentially obviating the need for intensive care. Further larger-scale prospective trials should be considered to further elucidate the role of this analgesia method. LEVEL OF EVIDENCE: Level III.

Factors Affecting Higher Readmission Rates and Costs in Pediatric Patients With Hirschsprung Disease.

BACKGROUND: Hirschsprung disease is a relatively uncommon disorder of the developing gastrointestinal tract that requires surgical intervention to restore long-term function. While readmission for Hirschsprung-related complications is a known concern in these patients, we sought to identify patient-level factors associated with a prolonged hospital stay, increased costs at the time of a pull-through operation, as well as the risk for all-cause inpatient readmission after surgery. We hypothesized that higher level of care requirement during the operative stay, age at operation, and length of stay (LOS) would portend increased readmissions and disease-related complications such as Hirschsprung-associated enterocolitis. METHODS: Data was obtained from the Pediatric Health Information System database on all Hirschprung patients who underwent a pull-through operation between 2004 and 2019. Regression analyses were performed on this cohort of 3345 patients. Multivariable regression models were utilized to analyze the key outcome variables of postoperative LOS and adjusted charges. RESULTS: Post-operative LOS was significantly increased by the presence of a surgical complication, congenital/genetic defect, or neurologic/neuromuscular defect. Increased LOS was also seen in Black patients. The cost of pull-through operations was significantly higher in patients admitted to the NICU and ICU during index hospitalization, with a cost increase of approximately $75,000 and $57,000 respectively. Presence of a surgical complication, comorbid congenital/genetic defect, and need for mechanical ventilation were associated with higher odds of inpatient readmission. CONCLUSION: The management of patients with Hirschsprung disease is longitudinal and complex. Identification of key patient metrics can aid clinicians in developing targeted care and education strategies to minimize readmission and excessive hospital charges.

The assessment of microbiome changes and fecal volatile organic compounds during experimental necrotizing enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) remains a devastating disease that affects the gastrointestinal tract of the preterm infant. Volatile organic compounds (VOCs) have emerged as a non-invasive biomarker in many diseases. We hypothesized that fecal VOC profiles would be significantly different between control and NEC pups in a NEC mouse model. METHODS: Experimental NEC was induced in five-day-old mice. Breastfed and formula-fed control groups were also studied. After four days, pups were euthanized and intestines were H&E stained and blindly scored. Stool microbiome analysis was performed via 16S rRNA sequencing. VOC analysis was assessed by the CyranoseⓇ 320 eNose device and p<0.05 was significant. RESULTS: NEC pups had severe intestinal injury when compared to controls. Microbiome analysis showed that both control groups had significantly higher microbial diversity and relative abundance of Lactobacillus than NEC, and lower relative abundance of Escherichia. Fecal VOC profile for NEC pups was significantly different from controls. CONCLUSIONS: Experimental NEC was associated with intestinal dysbiosis. Fecal VOC analysis by the CyranoseⓇ 320 eNose device can discriminate NEC pups from both breastfed and formula-fed controls. Further research is warranted to establish whether fecal VOCs can be used as a biomarker or predictive algorithm to diagnose NEC.

It's all in the milk: chondroitin sulfate as potential preventative therapy for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating condition affecting up to 5% of neonatal intensive care unit (NICU) admissions. Risk factors include preterm delivery, low birth weight, and antibiotic use. The pathogenesis is characterized by a combination of intestinal ischemia, necrosis of the bowel, reperfusion injury, and sepsis typically resulting in surgical resection of afflicted bowel. Targeted medical therapy remains elusive. Chondroitin sulfate (CS) holds the potential to prevent the onset of NEC through its anti-inflammatory properties and protective effect on the gut microbiome. The purpose of this review is to outline the many properties of CS to highlight its potential use in high-risk infants and attenuate the severity of NEC. The purpose of this review is to (1) discuss the interaction of CS with the infant microbiome, (2) review the anti-inflammatory properties of CS, and (3) postulate on the potential role of CS in preventing NEC. IMPACT: NEC is a costly medical burden in the United States. Breast milk is the best preventative measure for NEC, but not all infants in the NICU have access to breast milk. Novel therapies and diagnostic tools are needed for NEC. CS may be a potential therapy for NEC due to its potent anti-inflammatory properties. CS could be added to the formula in an attempt to mitigate breast milk disparities.

Angiogenesis: A Cellular Response to Traumatic Injury.

ABSTRACT: The development of new vasculature plays a significant role in a number of chronic disease states, including neoplasm growth, peripheral arterial disease, and coronary artery disease, among many others. Traumatic injury and hemorrhage, however, is an immediate, often dramatic pathophysiologic insult that can also necessitate neovascularization to promote healing. Traditional understanding of angiogenesis involved resident endothelial cells branching outward from localized niches in the periphery. Additionally, there are a small number of circulating endothelial progenitor cells that participate directly in the process of neovessel formation. The bone marrow stores a relatively small number of so-called pro-angiogenic hematopoietic progenitor cells-that is, progenitor cells of a hematopoietic potential that differentiate into key structural cells and stimulate or otherwise support local cell growth/differentiation at the site of angiogenesis. Following injury, a number of cytokines and intercellular processes are activated or modulated to promote development of new vasculature. These processes initiate and maintain a robust response to vascular insult, allowing new vessels to canalize and anastomose and provide timely oxygen delivering to healing tissue. Ultimately as we better understand the key players in the process of angiogenesis we can look to develop novel techniques to promote healing following injury.

The Assessment of Fecal Volatile Organic Compounds in Healthy Infants: Electronic Nose Device Predicts Patient Demographics and Microbial Enterotype.

BACKGROUND: The assessment of fecal volatile organic compounds (VOCs) has emerged as a noninvasive biomarker in many different pathologies. Before assessing whether VOCs can be used to diagnose intestinal diseases, including necrotizing enterocolitis (NEC), it is necessary to measure the impact of variable infant demographic factors on VOC signals. MATERIALS AND METHODS: Stool samples were collected from term infants at four hospitals in a large metropolitan area. Samples were heated, and fecal VOCs assessed by the Cyranose 320 Electronic Nose. Twenty-eight sensors were combined into an overall smellprint and were also assessed individually. 16s rRNA gene sequencing was used to categorize infant microbiomes. Smellprints were correlated to feeding type (formula versus breastmilk), sex, hospital of birth, and microbial enterotype. Overall smellprints were assessed by PERMANOVA with Euclidean distances, and individual sensors from each smellprint were assessed by Mann-Whitney U-tests. P < 0.05 was significant. RESULTS: Overall smellprints were significantly different according to diet. Individual sensors were significantly different according to sex and hospital of birth, but overall smellprints were not significantly different. Using a decision tree model, two individual sensors could reliably predict microbial enterotype. CONCLUSIONS: Assessment of fecal VOCs with an electronic nose is impacted by several demographic characteristics of infants and can be used to predict microbiome composition. Further studies are needed to design appropriate algorithms that are able to predict NEC based on fecal VOC profiles.