Bovine milk-derived exosomes attenuate NLRP3 inflammasome and NF-κB signaling in the lung during neonatal necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC), an inflammatory intestinal disease common in premature infants, has been associated with the development of lung damage. Toll-like receptor 4 has been shown to regulate inflammation in the NEC lungs, however, other important inflammatory mechanisms have not been thoroughly investigated. In addition, we reported that milk-derived exosomes were able to attenuate intestinal injury and inflammation in experimental NEC. This study aims to (i) investigate the role of the NLRP3 inflammasome and NF-κB pathway in regulating lung damage during experimental NEC; and (ii) evaluate the therapeutic potential of bovine milk exosomes in reducing lung inflammation and injury during NEC. METHODS: NEC was induced by gavage feeding of hyperosmolar formula, hypoxia, and lipopolysaccharide administration in neonatal mice from postnatal days 5-9. Exosomes were obtained by ultracentrifugation of bovine milk and administered during each formula feed. RESULTS: The lung of NEC pups showed increased inflammation, tissue damage, NLRP3 inflammasome expression, and NF-κB pathway activation, which were attenuated upon exosome administration. CONCLUSION: Our findings suggest that the lung undergoes significant inflammation and injury following experimental NEC which are attenuated by bovine milk-derived exosomes. This emphasizes the therapeutic potential of exosomes not just on the intestine but also on the lung.

Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy.

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

Activation of Wnt signaling by amniotic fluid stem cell-derived extracellular vesicles attenuates intestinal injury in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting preterm neonates and causing high morbidity, high mortality, and huge costs for the family and society. The treatment and the outcome of the disease have not changed in recent decades. Emerging evidence has shown that stimulating the Wnt/ß-catenin pathway and enhancing intestinal regeneration are beneficial in experimental NEC, and that they could potentially be used as a novel treatment. Amniotic fluid stem cells (AFSC) and AFSC-derived extracellular vesicles (EV) can be used to improve intestinal injury in experimental NEC. However, the mechanisms by which they affect the Wnt/ß-catenin pathway and intestinal regeneration are unknown. In our current study, we demonstrated that AFSC and EV attenuate NEC intestinal injury by activating the Wnt signaling pathway. AFSC and EV stimulate intestinal recovery from NEC by increasing cellular proliferation, reducing inflammation and ultimately regenerating a normal intestinal epithelium. EV administration has a rescuing effect on intestinal injury when given during NEC induction; however, it failed to prevent injury when given prior to NEC induction. AFSC-derived EV administration is thus a potential emergent novel treatment strategy for NEC.

Impaired Wnt/ß-catenin pathway leads to dysfunction of intestinal regeneration during necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by acute intestinal injury. Intestinal stem cell (ISC) renewal is required for gut regeneration in response to acute injury. The Wnt/ß-catenin pathway is essential for intestinal renewal and ISC maintenance. We found that ISC expression, Wnt activity and intestinal regeneration were all decreased in both mice with experimental NEC and in infants with acute active NEC. Moreover, intestinal organoids derived from NEC-injured intestine of both mice and humans failed to maintain proliferation and presented more differentiation. Administration of Wnt7b reversed these changes and promoted growth of intestinal organoids. Additionally, administration of exogenous Wnt7b rescued intestinal injury, restored ISC, and reestablished intestinal epithelial homeostasis in mice with NEC. Our findings demonstrate that during NEC, Wnt/ß-catenin signaling is decreased, ISC activity is impaired, and intestinal regeneration is defective. Administration of Wnt resulted in the maintenance of intestinal epithelial homeostasis and avoidance of NEC intestinal injury.

Bovine milk-derived exosomes enhance goblet cell activity and prevent the development of experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is characterized by intestinal injury and impaired mucin synthesis. We recently showed that breast milk exosomes from rodents promote intestinal cell viability, epithelial proliferation, and stem cell activity, but whether they also affect mucus production is unknown. Therefore, the aim of this study was to investigate the effects of bovine milk-derived exosomes on goblet cell expression in experimental NEC and delineate potential underlying mechanisms of action. Exosomes were isolated from bovine milk by ultracentrifugation and confirmed by Nanoparticle Tracking Analysis and through the detection of exosome membrane markers. To study the effect on mucin production, human colonic LS174T cells were cultured and exposed to exosomes. Compared to control, exosomes promoted goblet cell expression, as demonstrated by increased mucin production and relative expression levels of goblet cell expression markers trefoil factor 3 (TFF3) and mucin 2 (MUC2). In addition, exosome treatment enhanced the expression of glucose-regulated protein 94 (GRP94), the most abundant intraluminal endoplasmic reticulum (ER) chaperone protein that aids in protein synthesis. Furthermore, experimental NEC was induced in mouse pups by hyperosmolar formula feeding, lipopolysaccharide administration and hypoxia exposure on postnatal days 5-9. Milk exosomes were given with each gavage feed. NEC was associated with ileal morphological injury and reduction in MUC2+ goblet cells and GRP94+ cells per villus. Exosome administration to NEC pups prevented these changes. This research highlights the potential novel application of milk-derived exosomes in preventing the development of NEC in high-risk infants when breast milk is not available.

Neonatal intestinal injury induced by maternal separation: pathogenesis and pharmacological targets 1.

Maternal separation (MS) is a well-studied phenomenon thought to play a role in the pathogenesis of many diseases ranging from neuropsychiatric to early intestinal disorders such as necrotizing enterocolitis. The existing evidence suggests that MS initiates a variety of processes that in turn lead to early intestinal injury. Although there are many theories as to how MS alters normal physiological processes, the exact mechanism of action remains to be elucidated. This review aims to describe some of the pathological processes affecting the intestine that are caused by MS, including (i) brain-gut axis, (ii) intestinal epithelial barrier function, (iii) microbiome, (iv) oxidative stress and endoplasmic reticulum stress, and (v) gut inflammation.

Value of abdominal ultrasound in management of necrotizing enterocolitis: a systematic review and meta-analysis.

PURPOSE: Necrotizing enterocolitis (NEC) remains a life-threatening disease among infants in the NICU. Early diagnosis and careful monitoring are essential to improve outcomes. Abdominal ultrasound (AUS) seems a promising addition to current diagnostic modalities, but its clinical utility is uncertain. The aim of this study was to identify AUS features associated with definite NEC (i.e. Bell stage ≥ II), failed medical treatment, surgery, and death. METHODS: Embase, MEDLINE, Web of Science and CINAHL databases were searched for studies that addressed any NEC-related AUS feature in relation to any of the four outcomes. After critical appraisal of relevant study methods, meta-analyses were conducted using a random-effect model. RESULTS: 15 out of 1215 studies were included. All AUS features had sensitivities below 70% and specificities largely above 80% for diagnosing definite NEC; several AUS features were significantly associated with failed medical treatment and surgery. Substantial heterogeneity, poor reporting quality and uncertain risk of bias were found. CONCLUSIONS: While clear associations of AUS features with failed medical treatment exist and AUS may detect definite NEC, substantial heterogeneity, poor reporting quality and an uncertain risk of bias impair the use of AUS for clinical decision making. A prospective, well-designed validation study is needed.

Are prophylactic anti-reflux medications effective after esophageal atresia repair? Systematic review and meta-analysis.

PURPOSE: Gastroesophageal reflux after surgical repair of esophageal atresia (EA) can be associated with complications, such as esophageal stricture. Recent guidelines recommend prophylactic anti-reflux medication (PARM) after EA repair. However, the effectiveness of PARM is still unclear. The aim of this study was to review evidence surrounding the use of PARM in children operated for EA. METHODS: We performed a systematic review and meta-analysis. We searched Medline, EMBASE, and the Cochrane Databases from inception until the end of 2016 for comparative studies of PARM versus no PARM (control). Primary outcome was postoperative esophageal stricture. Quality of evidence was assessed using GRADE system. RESULTS: We identified four observational studies that focused on esophageal stricture as an outcome. A total of 362 patients were included in meta-analysis. There was no significant difference in esophageal stricture rates between PARM and control (OR = 1.14; 95% CI = 0.61-2.13; p = 0.68; I2 = 38%). The quality of the evidence was very low, due to lack of precision as a consequence of small study sizes. CONCLUSIONS: Our results indicate that PARM does not reduce the incidence of esophageal stricture after EA repair. Future well-controlled prospective studies are needed to obtain higher quality evidence.

Initiation of Enteral Feeding After Necrotizing Enterocolitis.

INTRODUCTION: Management of necrotizing enterocolitis (NEC) consists of cessation of enteral feeding, intravenous antibiotic administration, and supportive treatment. There is no evidence-based recommendation regarding when to restart feeding after a NEC episode. We performed a systematic review and meta-analysis to examine the effect of timing of enteral feeding reinitiation on NEC recurrence. METHODS: MEDLINE, Embase, Google scholar, and Cochrane databases were searched. Human studies evaluating enteral feeding timing with a primary outcome of NEC recurrence were included. A total of 2,257 titles or abstracts were screened, and 47 full-text articles were analyzed. A systematic review and meta-analysis comparing NEC recurrence and other associated outcomes between early (<5 days after NEC diagnosis) and delayed (>5 days) initiation of enteral feeding after NEC were performed according to the PRISMA statement. The meta-analysis data were analyzed using RevMan 5.3 to estimate odds ratios (ORs) with 95% confidence intervals (CIs). RESULTS: Two retrospective observational studies met the inclusion criteria, comprising 56 cases in which enteral feeding was started early and 35 cases of delayed enteral feeding initiation. There were no randomized controlled trials (RCTs). The recurrence rates of NEC were unchanged between early (5.4%) and delayed (8.6%) enteral feeding groups (pooled OR = 0.61; 95% CI: 0.12-3.16; p = 0.56; I2 = 0%). Catheter-related sepsis (pooled OR = 0.20; 95% CI: 0.01-3.29; p = 0.26; I2 = 67%) and post-NEC stricture (pooled OR = 0.28; 95% CI: 0.07-1.18; p = 0.08; I2 = 23%) rates were not different between early and delayed enteral feeding groups. CONCLUSION: Initiating early enteral feeding, within 5 days of NEC diagnosis, is not associated with adverse outcomes, including NEC recurrence. In addition, catheter-related sepsis and post-NEC stricture rates were unchanged between early and delayed enteral feeding groups after NEC. However, the quality of the evidence from the review of literature is suboptimal. A further RCT is needed to confirm these results.

Duhamel and Transanal Endorectal Pull-throughs for Hirschsprung' Disease: A Systematic Review and Meta-analysis.

AIM: The Duhamel pull-through and transanal endorectal pull-through (TEPT) are commonly used for the treatment of Hirschsprung's disease (HD). To date, there has been no meta-analysis evaluating postoperative outcomes following Duhamel pull-through and TEPT. The purpose of this meta-analysis was to compare patient outcome after Duhamel pull-through and TEPT for HD. MATERIALS AND METHODS: Original articles published between 1998 and 2016 were identified using the MEDLINE database. Studies comparing Duhamel pull-through and TEPT were included. Outcomes evaluated included incidence of postoperative constipation, incontinence/soiling, enterocolitis, anastomotic stricture, and leak. We analyzed dichotomous variables by estimating odds ratios (OR) with 95% confidence intervals (CI) and continuous variables using the weighted mean difference with 95% CI. The meta-analysis was done using RevMan 5.3. RESULT: There were no randomized controlled trials. Seven observational clinical studies were included, comprising 260 cases of Duhamel pull-through and 170 cases of TEPT. Anastomotic stricture (OR = 0.10; 95%CI 0.02-0.48; p = 0.004) was lower following Duhamel pull-through than TEPT. There were no significant differences in the incidence of postoperative incontinence/soiling and anastomotic leak. After TEPT, postoperative constipation seems to be lower and enterocolitis higher compared with those after Duhamel pull-through; however, these differences are not significant when the follow-up period is equal between groups. CONCLUSION: The Duhamel pull-through seems to be associated with lower incidence of anastomotic stricture compared with TEPT. The effects of the two analyzed operative techniques on constipation and enterocolitis remain unclear. The quality of evidence supporting the above findings is suboptimal, indicating the need for prospective studies.

Inhibition of corticotropin-releasing hormone receptor 1 and activation of receptor 2 protect against colonic injury and promote epithelium repair.

Maternal separation (MS) in neonates can lead to intestinal injury. MS in neonatal mice disrupts mucosal morphology, induces colonic inflammation and increases trans-cellular permeability. Several studies indicate that intestinal epithelial stem cells are capable of initiating gut repair in a variety of injury models but have not been reported in MS. The pathophysiology of MS-induced gut injury and subsequent repair remains unclear, but communication between the brain and gut contribute to MS-induced colonic injury. Corticotropin-releasing hormone (CRH) is one of the mediators involved in the brain-gut axis response to MS-induced damage. We investigated the roles of the CRH receptors, CRHR1 and CRHR2, in MS-induced intestinal injury and subsequent repair. To distinguish their specific roles in mucosal injury, we selectively blocked CRHR1 and CRHR2 with pharmacological antagonists. Our results show that in response to MS, CRHR1 mediates gut injury by promoting intestinal inflammation, increasing gut permeability, altering intestinal morphology, and modulating the intestinal microbiota. In contrast, CRHR2 activates intestinal stem cells and is important for gut repair. Thus, selectively blocking CRHR1 and promoting CRHR2 activity could prevent the development of intestinal injuries and enhance repair in the neonatal period when there is increased risk of intestinal injury such as necrotizing enterocolitis.

Breast milk-derived exosomes promote intestinal epithelial cell growth.

BACKGROUND: Breast milk administration prevents necrotizing enterocolitis (NEC). However, the mechanism remains unclear. Exosomes are cell-derived vesicles highly present in human milk and regulate intercellular signaling, inflammation, and immune response. We hypothesized that milk-derived exosomes beneficially affect intestinal epithelial cells. METHODS: Rat milk was collected, and exosomes were isolated using ExoQuick reagent and visualized by Nanoparticle Tracking Analysis. Protein was extracted from encapsulating exosomes, and concentration was measured. 2×104 intestinal epithelial cells (IEC-18) were treated for five hours with 0.5-µg/µl exosomes, an equal volume of exosome-free milk, or control solution (PBS). IEC-18 viability was measured using a colorimetric assay (MTT), and gene expression was analyzed by qRT-PCR. Data were compared using one-way ANOVA with Bonferroni post-test. RESULTS: Rat milk was collected, and exosome isolation was confirmed. Compared to control, treatment with exosomes significantly increased IEC viability, proliferation, and stem cell activity (all p<0.05). However, administration of exosome-free milk had less significant effects. CONCLUSIONS: Rat milk-derived exosomes promote IEC viability, enhance proliferation, and stimulate intestinal stem cell activity. These findings provide insight into the mechanism of action of breast milk in the intestines. Exosome administration is a promising prevention method for infants at risk of developing NEC when breastfeeding is not tolerated.

Gastric emptying is reduced in experimental NEC and correlates with the severity of intestinal damage.

PURPOSE: The aim of this study is to assess gastric emptying in experimental necrotizing enterocolitis (NEC) and its diagnostic significance using non-invasive ultrasound imaging. METHODS: Fourteen neonatal mice (C57BL/6) were randomized into two groups: NEC [n=10] and control [n=4]. NEC was induced by gavage feeding of hyperosmolar formula, hypoxia, and lipopolysaccharide between postnatal day 5 (P5) and 9 (P9). Stomach volume was measured using a 40-MHz ultrasound transducer on P5 and P9. Gastric residual volume was calculated in control mice from two measurements at 4h interval and in NEC mice from two measurements immediately after gavage feeding and 4h post-fasting. The distal ileum was harvested for histology and quantitative PCR analysis on P9. RESULTS: On P9, NEC mice had a greater gastric residual volume compared to control (p=0.002) indicating delay in gastric emptying. Positive correlations were found between gastric residual volume and both IL-6 mRNA expression level and histological bowel damage (p=0.035, p=0.012, respectively). CONCLUSIONS: During experimental NEC there is a delay in stomach emptying which is related to the severity of the disease. Ultrasound assessment of gastric emptying is a new non-invasive imaging modality that could be used to predict the severity of NEC.

Intestinal epithelial injury induced by maternal separation is protected by hydrogen sulfide.

PURPOSE: Oxidative stress has been implicated in the pathogenesis of various neonatal diseases involving the intestine. Hydrogen sulfide (H2S) has been shown to protect against oxidative stress. We hypothesized that administration of sodium hydrosulfide (NaHS), an H2S donor, to neonatal mice can decrease the intestinal epithelial injury associated with maternal separation (MS). METHODS: C57BL/6 mice received either intraperitoneal phosphate buffered saline (PBS; n=10) or NaHS (1mg/kg/day; n=10), followed by MS for 3h daily between postnatal day P5 and P9. Control neonatal mice were untreated and were not exposed to MS (n=10). Proximal colon was harvested and analyzed for crypt length, goblet cell number per crypt, oxidative stress and inflammation. Groups were compared using one-way ANOVA with Bonferroni post-test. RESULTS: Compared to controls, MS+PBS mice had shorter crypt lengths, fewer goblet cells per crypt, reduced glutathione peroxidase activity, increased expression of thiobarbituric acid reactive substances and inducible nitric oxide synthase mRNA, as well as increased IL-6, TNFα and myeloperoxidase. Administration of NaHS significantly counteracted these negative effects of MS. CONCLUSIONS: H2S protects the colon from the epithelial damage, oxidative stress and inflammation caused by maternal separation. This study provides insights on the pathogenesis of neonatal bowel diseases and indicates the potential for a pharmacological intervention to rescue the colonic epithelium. LEVEL OF EVIDENCE: n/a - animal and laboratory study.

Osmolality of enteral formula and severity of experimental necrotizing enterocolitis.

PURPOSE: Administration of hyperosmolar formula is regarded as a risk factor for the development of necrotizing enterocolitis (NEC). However, there are limited number of reports about the relationship between formula osmolality and NEC. The aim of this study is to evaluate the effects of formula concentration in an experimental model of NEC. METHODS: We studied experimental NEC in C57BL/6 mice. NEC was induced by giving hypoxia, gavage administration of lipopolysaccharide and gavage formula feeding from postnatal day 5-9. We used two types of formula: (1) hyperosmolar formula (HF): 15 g Similac + 75 ml Esbilac (849 mOsm/kg); (2) diluted formula (DF): dilute hyperosmolar formula with an equal amount of water (325 mOsm/kg). Controls were fed by the mother. On postnatal day 9, the ileum was harvested and evaluated for severity of mucosal injury (hematoxylin/eosin staining) and inflammation (PCR for IL6 and TNFα mRNA expression). RESULTS: The incidence of NEC was same in both HF and DF (80%). The intestinal inflammatory response was similar between HF and DF (IL6: p = 0.26, TNFα: p = 0.69). CONCLUSIONS: This study indicates the osmolality of enteral formula does not affect incidence of experimental NEC. This experimental study provides new insights into the relationship between formula feeding and NEC.