Children with Hirschsprung disease exhibited alterations in host-microbial co-metabolism after pull-through operation.

PURPOSE: This study aims to compare the fecal metabolome in post pull-through HD with and without HAEC patients and healthy young children using nuclear magnetic resonance (NMR) spectroscopy. METHODS: Fresh fecal samples were collected from children under 5 years of age in both post-pull-through HD patients and healthy Thai children. A total of 20 fecal samples were then analyzed using NMR spectroscopy. RESULTS: Thirty-four metabolites identified among HD and healthy children younger than 5 years were compared. HD samples demonstrated a significant decrease in acetoin, phenylacetylglutamine, and N-acetylornithine (corrected p value = 0.01, 0.04, and 0.004, respectively). Succinate and xylose significantly decreased in HD with HAEC group compared to HD without HAEC group (corrected p value = 0.04 and 0.02, respectively). Moreover, glutamine and glutamate metabolism, and alanine, aspartate, and glutamate metabolism were the significant pathways involved, with pathway impact 0.42 and 0.50, respectively (corrected p value = 0.02 and 0.04, respectively). CONCLUSION: Differences in class, quantity, and metabolism of protein and other metabolites in young children with HD after pull-through operation were identified. Most of the associated metabolic pathways were correlated with the amino acids metabolism, which is required to maintain intestinal integrity and function.

Development of liver inflammatory injury in biliary atresia: from basic to clinical research.

Biliary atresia (BA) is a severe cholangiopathy in infants. It is characterized by inflammatory fibro-obliteration of the intra- and extrahepatic bile ducts. Although the restoration of bile flow can be successful after Kasai operation, the rapid progression of liver fibrosis can continue, leading to cirrhosis. It is believed that the progression of liver fibrosis in BA is exacerbated by complicated mechanisms other than the consequence of bile duct obstruction. The fibrogenic cascade in BA liver can be divided into three stages, including liver inflammatory injury, myofibroblast activation, and fibrous scar formation. Recent studies have revealed that the activation of an immune response following bile duct injury plays an important role in promoting the inflammatory process, the releasing of inflammatory cytokines, and the development of fibrogenesis in BA liver. In this article, we summarized the evidence regarding liver inflammatory injury and the possible mechanisms that explain the rapid progression of liver fibrosis in BA.

Argon inhalation attenuates systemic inflammation and rescues lung architecture during experimental neonatal sepsis.

PURPOSE: Neonatal sepsis is a systemic inflammatory infection common in premature infants and a leading cause of mortality. Argon is an emerging interest in the field of noble gas therapy. Neonates with severe sepsis are frequently mechanically ventilated creating an opportunity for inhalation therapy. We aimed to investigate argon inhalation as a novel experimental therapy in neonatal sepsis. METHODS: Sepsis was established in C57BL/6 neonatal mice by a lipopolysaccharide intraperitoneal injection on postnatal day 9. Septic pup mice were exposed to room air as well as non-septic controls. In the argon group, septic pup mice were exposed to argon (70% Ar, 30% O2) for 6 h in a temperature-controlled environment. RESULTS: At 6 h, survival was significantly enhanced when septic mice received argon compared to septic controls. Serum profiles of cytokine release were significantly attenuated as well as lung architecture restored. CONCLUSIONS: Our findings suggest that argon inhalation as a novel treatment for neonatal sepsis, reducing mortality and counteracting the acute systemic inflammatory response in the blood and preserving the architecture of the lung. This research can contribute to a paradigm shift in the treatment and outcome of neonates with sepsis.

A comparison of surgical site infections in children after stoma reversal between purse-string and linear closure.

BACKGROUND: To reduce the surgical site infections (SSI), the purse-string closure technique has been widely performed and has also been recommended in adult stoma reversal. However, for children, some debate still exists. This study aims to compare the SSI rates in children between the purse-string and the linear for the skin closure of stoma reversal. METHODS: The data were collected from pediatric patients, who had undergone either purse-string or linear closure for elective surgery of stoma reversal from two university hospitals between January 2016 and December 2019. RESULTS: The purse-string and linear closure had been performed on 31 and 45 patients, respectively. At 30 days after surgery, three patients in the purse-string closure group had developed SSI compared to 14 patients in the linear closure group (9.7 vs. 31.1%, p = 0.028). Furthermore, there had been no significant difference in the overall post-operative complications. In multivariate analysis, the SSI had been significantly lower in patients with purse-string closure (OR 0.21, 95% CI 0.05-0.86, p = 0.029). CONCLUSION: By employing the purse-string closure technique for skin closure of stoma reversal, there had been a significantly lower SSI rate compared to linear closure with no difference in the length of hospital stay.

The intestinal injury caused by ischemia-reperfusion is attenuated by amniotic fluid stem cells via the release of tumor necrosis factor-stimulated gene 6 protein.

Ischemia/reperfusion (I/R) is implicated in the pathogenesis of various acute intestinal injuries. Amniotic fluid stem cells (AFSC) are beneficial in experimental intestinal diseases. Tumor necrosis factor-induced protein 6 (TSG-6) has been shown to exert anti-inflammatory effects. We aimed to investigate if AFSC secreted TSG-6 reduces inflammation and rescues intestinal I/R injury. The superior mesenteric artery of 3-week-old rats was occluded for 90 minutes and green fluorescent protein-labeled AFSC or recombinant TSG-6 was injected intravenously upon reperfusion. AFSC distribution was evaluated at 24, 48, and 72 hours after I/R. AFSC and TSG-6 effects on the intestine were assessed 48 hours postsurgery. Intestinal organoids were used to study the effects of TSG-6 after hypoxia-induced epithelial damage. After I/R-induced intestinal injury, AFSC migrated preferentially to the ileum, the primary site of injury, through blood circulation. Engrafted AFSC reduced ileum injury, inflammation, and oxidative stress. These AFSC-mediated beneficial effects were dependent on secretion of TSG-6. Administration of TSG-6 protected against hypoxia-induced epithelial damage in intestinal organoids. Finally, TSG-6 attenuated intestinal damage during I/R by suppressing genes involved in wound and injury pathways. This study indicates that AFSC or TSG-6 have the potential of rescuing the intestine from the damage caused by I/R.

Meso-Rex bypass versus portosystemic shunt for the management of extrahepatic portal vein obstruction in children: systematic review and meta-analysis.

PURPOSE: Extrahepatic portal vein obstruction (EHPVO) is a major cause of non-cirrhotic portal hypertension in children. Surgical procedures for EHPVO include portosystemic shunts (PSS) and meso-Rex bypass (MRB). We conducted a systematic review and meta-analysis to compare the effectiveness of MRB versus PSS in EHPVO patients. METHODS: A systematic literature search was performed using four databases. Articles reporting EHPVO and comparing patients who received MRB and PSS were included in the analysis. RESULTS: We retrieved 851 papers, of which five observational studies met the inclusion criteria. There was no difference in shunt complications, mortality, or gastrointestinal bleeding after surgery between MRB and PSS in the meta-analysis. MRB had increased shunt complications compared with PSS in the non-comparative studies. MRB had a potential advantage over PSS in long-term prognosis in one comparative study. Overall, the quality of the evidence was low. CONCLUSIONS: Based on available data, our meta-analysis indicates that MRB does not increase shunt complications, mortality, or gastrointestinal bleeding after surgery. The present study did not reveal superiority for either MRB or PSS. The paucity of well conducted trials in this area justifies future multicenter studies and studies that examine long-term outcomes.

Nasogastric decompression after intestinal surgery in children: a systematic review and meta-analysis.

PURPOSE: Postoperative nasogastric decompression has been routinely used after intestinal surgery. However, the role of nasogastric decompression in preventing postoperative complications and promoting the recovery of bowel function in children remains controversial. This systematic review aimed to assess whether routine nasogastric decompression is necessary after intestinal surgery in children. METHODS: A systematic review was conducted following the PRISMA guideline. Literature search was performed in electronic databases including PubMed, Embase, CENTRAL, and Web of science. Studies comparing outcomes between children who underwent intestinal surgery with postoperative nasogastric tube (NGT) placement (NGT group) and without postoperative NGT placement (no NGT group) were included. RESULTS: Six studies were eligible for inclusion criteria including two randomized controlled trials (RCT) and four comparative observational studies. The overall rate of postoperative anastomotic leak was 0.6% (1/179) in NGT group and 0.9% (2/223) in no NGT group. The overall rate of wound dehiscence was 2.4% (4/169) in NGT group and 1.6% (4/245) in no NGT group. Meta-analysis of two RCTs in children undergoing elective intestinal surgery showed significant increase of mild vomiting in no NGT group compared with NGT group (OR 3.54 95% CI 1.04, 11.99) but no significant difference in persistent vomiting requiring NGT reinsertion (OR 3.11 95% CI 0.47, 20.54), abdominal distension (OR 2.36 95% CI 0.34, 16.59), NGT reinsertion (OR 3.11 95% CI 0.47, 20.54), wound infection (OR 1.63 95% CI 0.49, 5.48) and time to return of bowel movement (MD - 0.14 95% CI - 0.45, 0.17). There was no incidence of anastomotic leak in these 2 RCTs. However, there was an incidence of NGT-related discomfort in NGT group, which ranged from 30 to 100% of children studied. CONCLUSION: Routine postoperative nasogastric decompression can be omitted in children undergoing intestinal surgery due to no benefit in preventing postoperative complications while increasing patient discomfort.

Amniotic fluid and breast milk: a rationale for breast milk stem cell therapy in neonatal diseases.

Amniotic fluid and breast milk play important roles in structural development throughout fetal growth and infancy. Given their significance in physical maturation, many studies have investigated the therapeutic and protective roles of amniotic fluid and breast milk in neonatal diseases. Of particular interest to researchers are stem cells found in the two fluids. These stem cells have been investigated due to their ability to self-replicate, differentiate, reduce tissue damage, and their expression of pluripotent markers. While amniotic fluid stem cells have received some attention regarding their ability to treat neonatal diseases, breast milk stem cells have not been investigated to the same extent given the recency of their discovery. The purpose of this review is to compare the functions of amniotic fluid, breast milk, and their stem cells to provide a rationale for the use of breast milk stem cells as a therapy for neonatal diseases. Breast milk stem cells present as an important tool for treating neonatal diseases given their ability to reduce inflammation and tissue damage, as well as their multilineage differentiation potential, easy accessibility, and ability to be used in disease modelling.

Intestinal organoids in infants and children.

Recent advances in culturing of intestinal stem cells and pluripotent stem cells have led to the development of intestinal organoids. These are self-organizing 3D structures, which recapitulate the characteristics and physiological features of in vivo intestinal epithelium. Intestinal organoids have allowed the development of novel in vitro models to study various gastrointestinal diseases expanding our understanding of the pathophysiology of diseases and leading to the development of innovative therapies. This article aims to summarize the current usage of intestinal organoids as a model of gastrointestinal diseases and the potential applications of intestinal organoids in infants and children. Intestinal organoids allow the study of intestinal epithelium responses to stress factors. Mimicking intestinal injury such as necrotizing enterocolitis, intestinal organoids increases the expression of pro-inflammatory cytokine genes and shows disruption of tight junctions after they are injured by lipopolysaccharide and hypoxia. In cystic fibrosis, intestinal organoids derived from rectal biopsies have provided benefits in genetic studies and development of novel therapeutic gene modulation. Transplantation of intestinal organoids via enema has been shown to rescue damaged colonic epithelium in mice. In addition, tissue-engineered small intestine derived from intestinal organoids have been successfully established providing a potential novel treatment and a new hope for children with short bowel syndrome.

Lysosomal overloading and necrotizing enterocolitis.

PURPOSE: Intestinal absorption in premature infants occurs via direct epithelial cellular endocytosis and degradation by intracellular lysosomes. Autophagy is a mechanism by which cytoplasmic organelles contribute to lysosomal degradation. However, excessive autophagy can lead to cell death. The purpose of this study was to investigate whether autophagy and endocytosis are present in the small intestinal mucosa during experimental necrotizing enterocolitis (NEC). METHODS: NEC was induced by gavage feeding of hyperosmolar formula, lipopolysaccharide and hypoxia between P5 and P9 (ethical approval 44032). Breastfed mice were used as control. Distal ileum was harvested on P5, P7 and P9 and analyzed for intestinal epithelial cellular morphology as well as autophagy/lysosomal activity, and cell death. Groups were compared using Student's t test. RESULTS: During NEC, giant lysosomes were present in the intestinal villi, with some exceeding their degradation ability leading to their rupture. The NEC group had significantly increased inflammation and autophagy activity, decreased lysosome activity, and increased apoptosis compared to control. CONCLUSIONS: NEC induction causes excessive autophagy and endocytosis leading to lysosomal overloading, lysosomal membrane permeabilization and rupture which results in cell death. These novel findings may help to clarify the pathogenesis of NEC. Reduction of lysosome overload and assisting in their degradation capability may reduce the burden of NEC.

A novel model of injured liver ductal organoids to investigate cholangiocyte apoptosis with relevance to biliary atresia.

PURPOSE: The fibrogenic process in cholangiopathic diseases such as biliary atresia (BA) involves bile duct injury and apoptosis of cholangiocytes, which leads to the progression of liver fibrosis into liver cirrhosis and can result in end-staged liver disease. Recent advances in the development of organoids or mini-organ structures have allowed us to create an ex vivo injury model of the bile duct that mimics bile duct injury in BA. The aim of this experimental study was to develop a novel model of injured intrahepatic cholangiocytes as this can be relevant to BA. Our new model is important for studying the pathophysiological response of bile ducts to injury and the role of cholangiocytes in initiating the fibrogenic cascade. In addition, it has the potential to be used as a tool for developing new treatment strategies for BA. METHODS: Liver ductal organoids were generated from the liver of healthy neonatal mouse pups. Intrahepatic bile duct fragments were isolated and cultured in Matrigel dome. Injury was induced in the organoids by administration of acetaminophen in culture medium. The organoids were then evaluated for fibrogenic cytokines expression, cell apoptosis marker and cell proliferation marker. RESULTS: Organoids generated from intrahepatic bile duct fragments organized themselves into single-layer epithelial spheroids with lumen on the inside mimicking in vivo bile ducts. After 24-h exposure to acetaminophen, cholangiocytes in the organoids responded to the injury by increasing expression of fibrogenic cytokines, transforming growth factor beta-1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). This fibrogenic response of injured organoids was associated with increased cholangiocyte apoptosis and decreased cholangiocyte proliferation. CONCLUSION: To our knowledge this is the first description of cholangiocyte injury in the organoids derived from intrahepatic bile ducts. Our injury model demonstrated that cholangiocyte apoptosis and its fibrogenic response may play a role in initiation of the fibrogenic process in cholangiopathic diseases such as BA. These findings are important for the development of novel therapy to reduce cholangiocyte apoptosis and to halt the early fibrogenic cascade in liver fibrogenesis. This novel injury model can prove very valuable for future research in biliary atresia.

Human breast milk exosomes attenuate intestinal damage.

BACKGROUND: Human breast milk (HBM), which contains an abundant supply of exosomes, is known to prevent necrotizing enterocolitis (NEC). Preterm infants are commonly given pasteurized donor milk when HBM is unavailable. However, pasteurization can disrupt its components. This study investigates the effects of both raw and pasteurized HBM-derived exosomes on intestinal inflammation. METHODS: HBM exosomes were isolated and characterized by positive CD63 and negative calnexin markers from western blot, nanoparticle tracking analysis and transmission electron microscopy. Mouse intestine organoids were established and treated with exosomes from raw or pasteurized HBM in healthy and injury conditions. Following ethical approval (#44032), mice pups were randomly assigned to (1) breastfed control; (2) NEC; (3) NEC receiving raw HBM exosomes; (4) NEC receiving pasteurized HBM exosomes. NEC was induced by hypoxia, gavage feeding and lipopolysaccharide (LPS). Ileum was evaluated. Data were analyzed using one-way ANOVA with Bonferroni post-test. RESULTS: Both raw and pasteurized HBM exosomes decreased inflammation in hypoxia and LPS-treated organoids compared to control. In vivo, NEC-induced mucosal injury, inflammation and mucous production were improved by raw and pasteurized HBM-derived exosomes. CONCLUSIONS: Exosomes derived from raw and pasteurized HBM equally reduced intestinal damage. Exosome administration in clinical practice requires further investigation.

The role of autophagy in intestinal epithelial injury.

PURPOSE: Autophagy is a natural mechanism aimed to degrade and recycle cellular components within cells. Previous studies reported that autophagy in the intestinal epithelium can be activated and that excessive autophagy can have negative consequences. However, the mechanism by which autophagy is regulated during intestinal epithelial injury remains unclear. This study aimed to investigate the mechanism of autophagy regulation during intestinal epithelial cells (IEC) injury. METHODS: Rat IEC18 were exposed to hypoxia and Lipopolysaccharide (LPS) (200 µg/ml) to induce injury. IEC18 were treated with autophagy initiation inhibitor, Wortmannin or with autophagy degradation inhibitor, Bafilomycin A1 were added for 24 h. We assessed the number and diameter of autophagic vacuoles, Cell viability, inflammation and apoptosis. RESULTS: Hypoxia and LPS administration increased the number and diameter of autophagic vacuoles in IEC18. Wortmannin administration reduced the number and diameter of autophagic vacuoles. On the contrary, Bafilomycin A1 administration increased the number of autophagic vacuoles. Cell viability increased following administration of Wortmannin and decreased following administration of Bafilomycin A1. CONCLUSIONS: We found that accumulation of autophagic vacuoles which characterize excessive or incomplete autophagy was detrimental to cell survival. This was shown by an increase in the number and size of the autophagic vacuoles with Bafilomycin A1treatment after hypoxia and LPS stressors relative to hypoxia and LPS alone. Conversely, there was a decrease in the number of autophagic vacuoles with Wortmannin treatment after hypoxia and LPS stressors relative to hypoxia and LPS alone. Therefore, reducing autophagosomes accumulation may represent a novel therapeutic strategy for intestinal injury.

Protective effects of vitamin D against injury in intestinal epithelium.

BACKGROUND: Vitamin D deficiency is associated with intestinal barrier dysfunction, which contributes to pathogenesis of acute intestinal injury in children. We aim to investigate the effects of vitamin D on intestinal injury in intestinal epithelial cells and organoids. METHODS: Lipopolysaccharide (LPS) was used to induce injury in intestinal epithelial cells (IEC-18) and organoids, and the effect of vitamin D was assessed. Cell viability was measured and inflammation cytokines TNFα and IL-8 were quantified. FITC-dextran 4 kDa (FD4) permeability was measured using Transwell while tight junction markers were assessed by immunofluorescence staining in IEC-18 and intestinal organoids. Data were compared using one-way ANOVA with Bonferroni post-test. RESULTS: IEC-18 viability was decreased by LPS treatment, but was prevented by vitamin D. The upregulation of inflammation was inhibited by vitamin D, which also decreased epithelium permeability. Vitamin D restored tight junction ZO-1 and claudin 2. In addition, vitamin D decreased TNFα expression and prevented the disruption of ZO-1 in injured organoids. CONCLUSIONS: Vitamin D rescued epithelial barrier function by improving permeability and restoring tight junctions, leading to decrease inflammation. This study confirms the protective effects of vitamin D, which could be used as a treatment strategy for infants at risk of developing intestinal injury.

Prognostic values of serum bilirubin at 7th day post-Kasai for survival with native livers in patients with biliary atresia.

BACKGROUND: Biliary atresia (BA) is a serious liver disease with uncertain prognosis. The objective of this study was to investigate prognostic values of the >20 % decrease in serum total bilirubin (TB) at 7th day post-op regarding early outcome and 5-year survival with native liver in BA. METHODS: Biliary atresia patients undergoing Kasai operation between 2000 and 2014 were reviewed. The ratio of serum TB at 7th day post-op to pre-op TB levels (TB7/TB0) was calculated for every patient. TB7/TB0 ratio of <0.8 indicated the >20 % decrease in serum TB. At 6th month following Kasai operation, outcome of BA patients were categorized into good outcome (TB < 2 mg % or clinically jaundice free) and poor outcome (TB > 2 mg % or clinically jaundice). For outcome analysis, logistic regression was used. For survival analysis, Cox regression was applied. RESULTS: There were 133 BA patients (M:F = 68:65) undergoing Kasai operation. Median age at surgery was 79 days. BA patients with TB7/TB0 ratio of <0.8 were found in 38 %. Outcome at 6-month post-op could be evaluated in 126 patients (good: poor = 68:58). The 1-, 3- and 5-year survival rates with native livers were 85, 70 and 65 %, respectively. The median overall survival with native livers was 164 months. Median follow-up time was 87 months. Logistic regression showed that gender and age at operation were not significant factors impacting on early outcome (p > 0.05). However, TB7/TB0 ratio of <0.8 was an independent factor for good outcome (Odds ratio = 3.0, p = 0.006). Cox regression analysis demonstrated that 5-year survival rate was significantly correlated with TB7/TB0 ratio of <0.8 (HR = 0.46, 95 % CI 0.23-0.91, p = 0.025) and outcome at 6th month post-op (HR = 0.05, 95 % CI 0.01-0.15, p < 0.001). CONCLUSIONS: The >20 % decrease in serum TB at 7th day post-Kasai is a predictor for good outcome. BA patients with TB7/TB0 of <0.8 had 5-year survival with native livers significantly higher than those with the ratio of >0.8.

Family care reduces the incidence of neonatal sepsis: A systematic review and meta-analysis.

Purpose: Family-involved care in the neonatal intensive care unit (NICU) helps to alleviate neonatal anxiety and promotes breastmilk intake, body growth and neurological development, but its effect on reducing the incidence of neonatal sepsis is not known. We conducted a systematic review and meta-analysis of randomized controlled trials (RCT) to evaluate whether neonates receiving family care have a lower incidence of neonatal sepsis compared to neonates receiving standard NICU care. Methods: MEDLINE, Embase, Web of Science, and CENTRAL were searched for RCTs that compared preterm neonates receiving family care vs. standard NICU care. From 126 articles that were identified and screened, 34 full-text articles were assessed for eligibility, and 5 RCTs were included. The primary outcome was the development of sepsis. The RevMan 5.4 software was used to conduct the Meta-analysis. Results: The metanalysis, based on 5 RCTs demonstrated that neonates receiving family-involved care had significantly lower incidence of sepsis (12.0% vs. 16.3%), increased body weight, and reduced length of hospital stay compared to those receiving standard NICU care. Conclusion: This study suggests that family-involved care in NICU can (i) reduce the incidence of neonatal sepsis, (ii) improve growth, and (iii) reduce the length of hospital stay. This study highlights the need for evaluating whether family-involved care improves other neonatal outcomes.

Human amniotic fluid stem cells attenuate cholangiocyte apoptosis in a bile duct injury model of liver ductal organoids.

PURPOSE: Biliary atresia (BA) is a fibro-obliterative cholangiopathy that involves both extrahepatic and intrahepatic bile ducts in infants. Cholangiocyte apoptosis has an influence on the fibrogenesis process of bile ducts and the progression of liver fibrosis in BA. Human amniotic fluid stem cells (hAFSCs) are multipotent cells that have ability to inhibit cell apoptosis. We aimed to investigate whether hAFSCs have the potential to attenuate cholangiocyte apoptosis and injury induced fibrogenic response in our ex vivo bile duct injury model of liver ductal organoids. METHODS: The anti-apoptotic effect of hAFSCs was tested in the acetaminophen-induced injury model of neonatal mouse liver ductal organoids (AUP #42681) by using direct and indirect co-culture systems. Cell apoptosis and proliferation were evaluated by immunofluorescent staining. Expression of fibrogenic cytokines was analyzed by RT-qPCR. Data were compared using one-way ANOVA with post hoc test. RESULTS: In our injury model, liver ductal organoids that were treated with hAFSCs in both direct and indirect co-culture systems had a significantly smaller number of apoptotic cholangiocytes and decreased expression of fibrogenic cytokines, transforming growth factor beta-1 (TGF-ß1) and platelet-derived growth factor-BB (PDGF-BB). Moreover, hAFSCs increased cholangiocyte proliferation in injured organoids. CONCLUSION: hAFSCs have the ability to protect the organoids from injury by decreasing cholangiocyte apoptosis and promoting cholangiocyte proliferation. This protective ability of hAFSCs leads to inhibition of the fibrogenic response in the injured organoids. hAFSCs have high therapeutic potential to attenuate liver fibrogenesis in cholangiopathic diseases such as BA.

Remote ischemic conditioning counteracts the intestinal damage of necrotizing enterocolitis by improving intestinal microcirculation.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants with high mortality rate, indicating the need for precision treatment. NEC is characterized by intestinal inflammation and ischemia, as well derangements in intestinal microcirculation. Remote ischemic conditioning (RIC) has emerged as a promising tool in protecting distant organs against ischemia-induced damage. However, the effectiveness of RIC against NEC is unknown. To address this gap, we aimed to determine the efficacy and mechanism of action of RIC in experimental NEC. NEC was induced in mouse pups between postnatal day (P) 5 and 9. RIC was applied through intermittent occlusion of hind limb blood flow. RIC, when administered in the early stages of disease progression, decreases intestinal injury and prolongs survival. The mechanism of action of RIC involves increasing intestinal perfusion through vasodilation mediated by nitric oxide and hydrogen sulfide. RIC is a viable and non-invasive treatment strategy for NEC.