Necrotizing enterocolitis: recent advances in treatment with translational potential.

Necrotizing enterocolitis (NEC) is one of the most prevalent and devastating gastrointestinal disorders in neonates. Despite advances in neonatal care, the incidence and mortality due to NEC remain high, highlighting the need to devise novel treatments for this disease. There have been a number of recent advancements in therapeutic approaches for the treatment of NEC; these involve remote ischemic conditioning (RIC), stem cell therapy, breast milk components (human milk oligosaccharides, exosomes, lactoferrin), fecal microbiota transplantation, and immunotherapy. This review summarizes the most recent advances in NEC treatment currently underway as well as their applicability and associated challenges and limitations, with the aim to provide new insight into the paradigm of care for NEC worldwide.

Amniotic fluid stem cell administration can prevent epithelial injury from necrotizing enterocolitis.

BACKGROUND: Stem cell therapy has been proven to rescue intestinal injury and stimulate intestinal regeneration in necrotizing enterocolitis (NEC). Specifically, stem cells derived from amniotic fluid (AFSCs) and mesenchymal stem cells (MSCs) derived from bone marrow have shown promising results in the treatment of experimental NEC. This study aims to examine the effects of AFSCs and MSCs on the prevention of intestinal injury during experimental NEC. METHODS: Supernatants from AFSC and MSC cultures were collected to perform proteomic analysis. Prior to NEC induction, mice received intraperitoneal injections of phosphate-buffered saline (PBS), 2 × 106 AFSCs, or 2 × 106 MSCs. RESULTS: We found that AFSCs grew faster than MSCs. Proteomic analysis indicated that AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. Administering AFSCs before NEC induction decreased NEC severity and mucosal inflammation. Intestinal proliferation and endogenous stem cell activation were increased after AFSC administration. However, administering MSCs before NEC induction had no beneficial effects. CONCLUSIONS: This study demonstrated that AFSCs and MSCs have different protein release profiles. AFSCs can potentially be used as a preventative strategy for neonates at risk of NEC, while MSCs cannot be used. IMPACT: AFSCs and MSCs have distinct protein secretory profiles, and AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. AFSCs are unique in transiently enhancing healthy intestinal epithelial cell growth, which offers protection against the development of experimental NEC. The prevention of NEC via the administration of AFSCs should be evaluated in infants at great risk of developing NEC or in infants with early signs of NEC.

Remote ischemic conditioning causes CD4 T cells shift towards reduced cell-mediated inflammation.

PURPOSE: Necrotizing enterocolitis (NEC) is a gastrointestinal disease in neonates that is associated with immune-mediated intestinal inflammation. Remote ischemic conditioning (RIC) applied to a limb has been shown to be protective against experimental NEC. In this study, we explore the immune cell-mediated response involved in NEC and the immunomodulatory effects of RIC in an experimental mouse model of the disease. METHODS: NEC was induced in C57BL/6 mice (ethical approval #58119) pups on postnatal day5 (p5) using gavage hyperosmolar formula, lipopolysaccharide, and hypoxia. RIC consisted of 4 cycles of 5 min ischemia followed by 5 min reperfusion of the right hindlimb during NEC induction on p6 and p8. Breastfed mice were used as control. The mice were sacrificed on p9, with ileal tissue evaluated for inflammatory cytokines and by characterization of T-cell populations. RESULTS: NEC mice had increased number of CD4+ cells indicating an accumulation of T-cells in the mesenchyme of the NEC ileum. Compared to control, NEC pups had upregulated expression pro-inflammatory cytokines (GATA3, IFNγ, IL1ß, IL6, IL17, IL22, and TNFα) and reduced anti-inflammatory cytokine (TGFß). In NEC, there was also a shift in the balance of Treg/Th17 cells towards Th17. Compared to NEC alone, RIC during the course of NEC resulted in reduction of pro-inflammatory cytokines (GATA3, IFNγ, IL1ß, IL6, IL17, IL22, and TNFα), increase in anti-inflammatory cytokine TGFß and concomitant shift back of Th17 cells towards Treg cells. CONCLUSION: In experimental NEC, remote ischemic conditioning reduces the production of pro-inflammatory markers and increases the production of anti-inflammatory markers. In addition, during NEC, RIC reverses the imbalance of Treg/Th17 providing support for its effect on cell-mediated inflammation. RIC is a non-invasive physical maneuver that can have a significant beneficial effect in reducing the inflammation seen in NEC.

Remote ischemic conditioning in necrotizing enterocolitis: study protocol of a multi-center phase II feasibility randomized controlled trial.

PURPOSE: Remote ischemic conditioning (RIC) is a maneuver involving brief cycles of ischemia reperfusion in an individual's limb. In the early stage of experimental NEC, RIC decreased intestinal injury and prolonged survival by counteracting the derangements in intestinal microcirculation. A single-center phase I study demonstrated that the performance of RIC was safe in neonates with NEC. The aim of this phase II RCT was to evaluate the safety and feasibility of RIC, to identify challenges in recruitment, retainment, and to inform a phase III RCT to evaluate efficacy. METHODS: RIC will be performed by trained research personnel and will consist of four cycles of limb ischemia (4-min via cuff inflation) followed by reperfusion (4-min via cuff deflation), repeated on two consecutive days post randomization. The primary endpoint of this RCT is feasibility and acceptability of recruiting and randomizing neonates within 24 h from NEC diagnosis as well as masking and completing the RIC intervention. RESULTS: We created a novel international consortium for this trial and created a consensus on the diagnostic criteria for NEC and protocol for the trial. The phase II multicenter-masked feasibility RCT will be conducted at 12 centers in Canada, USA, Sweden, The Netherlands, UK, and Spain. The inclusion criteria are: gestational age < 33 weeks, weight ≥ 750 g, NEC receiving medical treatment, and diagnosis established within previous 24 h. Neonates will be randomized to RIC (intervention) or no-RIC (control) and will continue to receive standard management of NEC. We expect to recruit and randomize 40% of eligible patients in the collaborating centers (78 patients; 39/arm) in 30 months. Bayesian methods will be used to combine uninformative prior distributions with the corresponding observed proportions from this trial to determine posterior distributions for parameters of feasibility. CONCLUSIONS: The newly established NEC consortium has generated novel data on NEC diagnosis and defined the feasibility parameters for the introduction of a novel treatment in NEC. This phase II RCT will inform a future phase III RCT to evaluate the efficacy and safety of RIC in early-stage NEC.

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.

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.

The effect of pre- and post-remote ischemic conditioning reduces the injury associated with intestinal ischemia/reperfusion.

PURPOSE: Midgut volvulus is associated with intestinal ischemia/reperfusion (IR) injury and can progress to severe intestinal damage. Remote ischemic conditioning (RIC) reduces IR-induced injury in distant organs. The aim of this study was to investigate whether RIC protects the intestine from IR injury. METHODS: We investigated intestinal IR injury in 3 weeks old SD rats. Animals underwent: (i) sham laparotomy, (ii) intestinal IR injury, (iii) intestinal IR + RIC during ischemia, or (iv) intestinal IR + RIC after reperfusion. Intestinal IR injury was achieved by 45 min occlusion of superior mesenteric artery followed by de-occlusion. RIC was administered via four cycles of 5 min of hind limb ischemia followed by 5 min reperfusion. Animals were sacrificed 24 h after surgery and the ileum was harvested for evaluation. RESULTS: Intestinal injury was present after IR. However, this injury was reduced in both IR + RIC groups. Expression of inflammatory cytokine IL6 was lower in IR + RIC groups compared to IR alone. Carbonyl protein was also significantly lower in IR + RIC compared to IR, indicating lower oxidative stress in both IR + RIC groups. CONCLUSION: Remote ischemic conditioning attenuated intestinal injury, inflammation, and oxidative stress in experimental intestinal ischemia/reperfusion injury. Remote ischemic conditioning may be useful in children with midgut volvulus to reduce the intestinal injury. LEVEL OF EVIDENCE: Experimental study. TYPE OF STUDY: Animal experiment.

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.

Evidence-based advances in minimally invasive surgery in infants with congenital gastrointestinal anomalies: a narrative review.

Background and Objective: Minimally invasive surgery (MIS) has been widely utilized to manage congenital gastrointestinal (GI) anomalies in children during the last two decades. Currently, MIS has a proven track-record for its feasibility and provides multiple benefits including better cosmesis, less trauma, and faster recovery in neonates and infants. However, it remains controversial whether MIS provides better definitive outcomes in pediatric patients with GI anomalies, especially among neonates. We aim to review the recent developments of MIS in infants with GI defects, assisting surgeons in making decisions and improving patient outcomes. Methods: A comprehensive literature search of PubMed and Web of Science's core collection was performed using terms of MIS techniques and congenital GI anomalies. Key Content and Findings: This review summarizes recent evidence-based advances of MIS in infants with congenital GI defects and potential future strategies based on evidence. Better cosmetic results, less postoperative pain, and an accelerated recovery have been shown to be common advantages of MIS relative to open approaches. Technical hurdles and metabolic disturbance were reported to be the main reasons for the decisions of open approach. Conclusions: Advanced techniques of MIS have made more precise manipulations and better outcomes possible, even for newborns. At the same time, surgeons should not be afraid to use an open approach in certain circumstances due to technical limitations or patient tolerance. The difficulty infants face in expressing their true feelings underscores the need for systematic and objective assessment tools to evaluate surgical outcomes.

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.

Remote ischemic conditioning in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disorder, most prevalent in premature infants, and associated with a high mortality rate that has remained unchanged in the past two decades. NEC is characterized by inflammation, ischemia, and impaired microcirculation in the intestine. Preclinical studies by our group have led to the discovery of remote ischemic conditioning (RIC) as a promising non-invasive intervention in protecting the intestine against ischemia-induced damage during early-stage NEC. RIC involves the administration of brief reversible cycles of ischemia and reperfusion in a limb (similar to taking standard blood pressure measurement) which activate endogenous protective signaling pathways that are conveyed to distant organs such as the intestine. RIC targets the intestinal microcirculation and by improving blood flow to the intestine, reduces the intestinal damage of experimental NEC and prolongs survival. A recent Phase I safety study by our group demonstrated that RIC was safe in preterm infants with NEC. A phase II feasibility randomized controlled trial involving 12 centers in 6 countries is currently underway, to investigate the feasibility of RIC as a treatment for early-stage NEC in preterm neonates. This review provides a brief background on RIC as a therapeutic strategy and summarizes the progression of RIC as a treatment for NEC from preclinical investigation to clinical evaluation.

Remote ischaemic conditioning in necrotising enterocolitis: a phase I feasibility and safety study.

OBJECTIVE: Remote ischaemic conditioning (RIC) improves the outcome of experimental necrotising enterocolitis (NEC) by preserving intestinal microcirculation. The feasibility and safety of RIC in preterm infants with NEC are unknown. The study aimed to assess the feasibility and safety of RIC in preterm infants with suspected or confirmed NEC. DESIGN: Phase I non-randomised pilot study conducted in three steps: step A to determine the safe duration of limb ischaemia (up to 4 min); step B to assess the safety of 4 repeated cycles of ischaemia-reperfusion at the maximum tolerated duration of ischaemia determined in step A; step C to assess the safety of applying 4 cycles of ischaemia-reperfusion on two consecutive days. SETTING: Level III neonatal intensive care unit, The Hospital for Sick Children (Toronto, Canada). PATIENTS: Fifteen preterm infants born between 22 and 33 weeks gestational age. INTERVENTION: Four cycles of ischaemia (varying duration) applied to the limb via a manual sphygmomanometer, followed by reperfusion (4 min) and rest (5 min), repeated on two consecutive days. OUTCOMES: The primary outcomes were (1) feasibility defined as RIC being performed as planned in the protocol, and (2) safety defined as perfusion returning to baseline within 4 min after cuff deflation. RESULTS: Four cycles/day of limb ischaemia (4 min) followed by reperfusion (4 min) and a 5 min gap, repeated on two consecutive days was feasible and safe in all neonates with suspected or confirmed NEC. CONCLUSIONS: This study is pivotal for designing a future randomised controlled trial to assess the efficacy of RIC in preterm infants with NEC. TRIAL REGISTRATION NUMBER: NCT03860701.

Design, synthesis, and cytotoxicity evaluation of novel indole-acylhydrazone derivatives of 4-pyridinone as potential histone deacetylase-2 inhibitors.

Background and purpose: Histone deacetylation is one of the essential cellular pathways in the growth and spread of cancer, so the design of histone deacetylase (HDAC) inhibitors as anticancer agents is of great importance in pharmaceutical chemistry. Here, a series of indole acylhydrazone derivatives of 4-pyridone have been introduced as potential histone deacetylase inhibitors. Experimental approach: Seven indole-acylhydrazone-pyridinone derivatives were synthesized via simple, straightforward chemical procedures. The molecular docking studies were accomplished on HDAC2 compared to panobinostat. The cytotoxicity of all derivatives was studied on MCF-7 and MDA-MB-231 breast cancer cell lines by MTT assay. Findings / Results: Molecular docking studies supported excellent fitting to the HADC2 active site with binding energies in the range of -10 Kcal/mol for all derivatives. All compounds were tested for their cytotoxicity against MCF-7 and MDA-MB-231 cell lines; derivatives A, B, F, and G were the best candidates. The half-maximal inhibitory concentration (IC50) values on MCF-7 were below 25 mg/mL and much lower than those obtained on the MDA-MB-231 cell line. Conclusion and implications: The derivatives showed selectivity toward the MCF-7 cell line, probably due to the higher HDAC expression in the MCF-7 cell line. In this regard, debenzylated derivatives F and G showed slightly better cytotoxicity, which should be more studied in the future. Derivatives A, B, F, and G were promising for future enzymatic studies.

A Real Saline Challenge: Diagnosing Primary Aldosteronism in the Setting of Chronic Kidney Disease.

Primary aldosteronism (PA) is the most common cause of secondary hypertension but remains largely undiagnosed. Chronic kidney disease (CKD) complicates the diagnosis of PA by affecting the biochemical screening evaluation and confirmatory testing, and by increasing the complication rate of adrenal venous sampling (AVS). To raise clinician awareness of the challenges of PA diagnosis in CKD, we present an illustrative case with subsequent review of the literature and discuss some recent developments in PA diagnostic strategies particularly applicable to the CKD population. A 67-year-old man with stage IIIb CKD was suspected of having PA due to treatment with 6 antihypertensive agents and the presence of intermittent hypokalemia. He had a positive biochemical screen for PA, and AVS demonstrated unilateral aldosterone excess. Subsequently, unilateral adrenalectomy resolved his PA, eliminating the patient's hypokalemia and improving his blood pressure. A MEDLINE literature search revealed 10 studies totaling 11 cases (including our own) of PA diagnosed in the setting of CKD. For each case, the clinical presentation, biochemical data, results of cross-sectional imaging, AVS details, and clinical response to surgery or medical therapy were characterized. The optimal strategy for the diagnosis and management of PA patients with CKD is not known. Although CKD patients often receive screening and subtype testing for PA similar to non-CKD patients, there are challenges in the interpretation of these tests. Novel strategies may include less invasive subtype testing or empiric treatment with mineralocorticoid receptor antagonists but additional studies are necessary.

Dual treatment of acromegaly and hormone-receptor-positive breast cancer with tamoxifen: a case report.

BACKGROUND: Adjuvant endocrine therapy is recommended for the treatment of hormone-receptor-positive breast cancer. Aromatase inhibitors are associated with significant musculoskeletal adverse effects, likely through growth hormone/insulin-like growth factor 1 modulation, while tamoxifen reduces insulin-like growth factor 1 production. We describe the case of a patient who was treated successfully with tamoxifen for her hormone-receptor-positive breast cancer and acromegaly. CASE PRESENTATION: A 57-year old White female with hormone-receptor-positive breast cancer was diagnosed with acromegaly. She received adjuvant endocrine therapy with anastrozole but could not tolerate this medication because of severe arthralgia, so she was switched to tamoxifen. Shortly after starting tamoxifen, the patient's musculoskeletal symptoms resolved and her insulin-like growth factor 1 levels normalized. She has remained in remission of her acromegaly and breast cancer since initiating tamoxifen. CONCLUSION: This case highlights the dual benefit of tamoxifen therapy in the treatment of hormone-receptor-positive breast cancer and acromegaly. Unlike anastrozole, tamoxifen has the benefit of lowering insulin-like growth factor 1 levels, which underscores its advantage in reducing adverse musculoskeletal symptoms during the treatment of hormone-receptor-positive breast cancer. We offer the first reported use of tamoxifen monotherapy for the successful treatment of acromegaly and hormone-receptor-positive breast cancer. While tamoxifen may offer an additional, oral option for acromegaly patients who do not respond to or tolerate conventional growth-hormone-lowering therapy, additional studies are necessary.

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.

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.

Treatment of necrotizing enterocolitis by conditioned medium derived from human amniotic fluid stem cells.

PURPOSE: Necrotizing enterocolitis (NEC) is one of the most distressing gastrointestinal emergencies affecting neonates. Amniotic fluid stem cells (AFSC) improve intestinal injury and survival in experimental NEC but are difficult to administer. In this study, we evaluated whether conditioned medium (CM) derived from human AFSC have protective effects. METHODS: Three groups of C57BL/6 mice were studied: (i) breast-fed mice as control; (ii) experimental NEC mice receiving PBS; and (iii) experimental NEC mice receiving CM. NEC was induced between post-natal days P5 through P9 via: (A) gavage feeding of hyperosmolar formula four-time a day; (B) 10 minutes hypoxia prior to feeds; and (C) lipopolysaccharide administration on P6 and P7. Intra-peritoneal injections of either PBS or CM were given on P6 and P7. All mice were sacrificed on P9 and terminal ileum were harvested for analyses. RESULTS: CM treatment increased survival and reduced intestinal damage, decreased mucosal inflammation (IL-6; TNF-α), neutrophil infiltration (MPO), and apoptosis (CC3), and also restored angiogenesis (VEGF) in the ileum. Additionally, CM treated mice had increased levels of epithelial proliferation (Ki67) and stem cell activity (Olfm4; Lgr5) compared to NEC+PBS mice, showing restored intestinal regeneration and recovery during NEC induction. CM proteomic analysis of CM content identified peptides that regulated immune and stem cell activity. CONCLUSIONS: CM derived from human AFSC administered in experimental NEC exhibited various benefits including reduced intestinal injury and inflammation, increased enterocyte proliferation, and restored intestinal stem cell activity. This study provides the scientific basis for the use of CM derived from AFSC in neonates with NEC.

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.