Necrotizing Enterocolitis Model Research Articles

Parthanatos initiated by ROS-induced DNA damage is involved in intestinal epithelial injury during necrotizing enterocolitis

Necrotizing enterocolitis (NEC) involves intestinal epithelial damage and inflammatory response and is associated with high morbidity and mortality in infants. To improve therapeutic prospects, elucidating underlying molecular mechanisms of intestinal epithelial damage during NEC is of the essence. Poly (ADP-ribose) polymerase 1 (PARP1)-dependent parthanatos is a programmed inflammatory cell death. In the present study, the presence of parthanatos-associated proteins PARP1 and poly (ADP-ribose) (PAR), along with high expression of DNA damage-associated biomarkers, 8-hydroxy-2’-deoxyguanosine (8-OHdG) and phosphorylation of histone H2AX (γH2AX), were discovered in the intestinal tissues of NEC infants. Additionally, the upregulated expression of PARP1 and PAR in NEC intestinal tissues correlated distinctly with clinical indices indicative of NEC incidence and severity. Furthermore, we demonstrated that inhibiting the expression of parthanatos-associated proteins, by either pharmacological blockage using 3-aminobenzamide (3-AB), an inhibitor of PARP1, or genetic knockout using Parp1-deficient mice, resulted in substantial improvements in both histopathological severity scores associated with intestinal injury and inflammatory reactions. Moreover, in an in vitro NEC model, reactive oxygen species (ROS)-induced DNA damage promoted the formation of PAR and nuclear translocation of apoptosis-inducing factor (AIF), thus activating PARP1-dependent parthanatos in Caco-2 cells and human intestinal organoids. Our work verifies a previously unexplored role for parthanatos in intestinal epithelial damage during NEC and suggests that inhibition of parthanatos may serve as a potential therapeutic strategy for intervention of NEC.

N-Acetylcysteine Alleviates Necrotizing Enterocolitis by Depressing SESN2 Expression to Inhibit Ferroptosis in Intestinal Epithelial Cells.

Abstract-Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease in neonates, and effective strategies to prevent and treat NEC are still lacking. Studies have shown that N-acetylcysteine (NAC) has protective effects against NEC, however, the specific mechanism underlying its effects on intestinal functions remains unclear. Recently, NAC has been shown to suppress ferroptosis in many diseases, while it is unclear whether the beneficial effects of NAC on NEC are related to ferroptosis. In this study, we revealed that ferroptosis was significantly induced in intestinal samples from infants with NEC. NAC alleviated intestinal inflammation, barrier damage and ferroptosis in multifactorial NEC models in vivo and in vitro. Sestrin2 (SESN2) was identified as an important mediator of NAC-induced ferroptosis resistance in intestinal epithelial cells. Furthermore, SESN2 knockdown inhibited the inflammatory response, alleviated barrier damage and ferroptosis in intestinal epithelial cells and enhanced the protective effects of NAC to a certain extent. Conversely, cells overexpressing SESN2 showed the opposite changes. In summary, our study demonstrated that NAC attenuates NEC progression by decreasing SESN2 expression to inhibit ferroptosis in intestinal epithelial cells, suggesting that NAC might be an effective clinical treatment for NEC.

Photoacoustic imaging for non-invasive assessment of biomarkers of intestinal injury in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an often-lethal disease of the premature infant intestinal tract, exacerbated by significant diagnostic difficulties. In NEC, the intestine exhibits hypoperfusion and dysmotility, contributing to disease pathogenesis. However, these features cannot be accurately and quantitively assessed with current imaging modalities. We have previously demonstrated the ability of photoacoustic imaging (PAI) to non-invasively assess intestinal tissue oxygenation and motility in a healthy neonatal rat model. In this first-in-disease application, we evaluated NEC using PAI to assess intestinal health biomarkers in an experimental model of NEC. NEC was induced in neonatal rats from birth to 4-days. Healthy breastfed (BF) and NEC rat pups were imaged at 2- and 4-days. Intestinal tissue oxygen saturation was measured with PAI, and NEC pups showed significant decreases at 2- and 4-days. Ultrasound and PAI cine recordings were used to capture intestinal peristalsis and contrast agent transit within the intestine. Intestinal motility, assessed using computational intestinal deformation analysis, demonstrated significant reductions in both early and established NEC. NEC damage was confirmed with histology and dysmotility was confirmed by small intestinal transit assay. This preclinical study presents PAI as an emerging diagnostic imaging modality for intestinal disease assessment in premature infants. Necrotizing enterocolitis (NEC) is a devastating intestinal disease affecting premature infants with significant mortality. NEC presents significant clinical diagnostic difficulties, with limited diagnostic confidence complicating timely and effective interventional efforts. This study is an important foundational first-in-disease preclinical study that establishes the utility for PAI to detect changes in intestinal tissue oxygenation and intestinal motility with NEC disease induction and progression. This study demonstrates the feasibility and exceptional promise for the use of PAI to non-invasively assess oxygenation and motility in the healthy and diseased infant intestine.

Extracellular Nicotinamide Phosphoribosyltransferase Is a Therapeutic Target in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of prematurity. Postulated mechanisms leading to inflammatory necrosis of the ileum and colon include activation of the pathogen recognition receptor Toll-like receptor 4 (TLR4) and decreased levels of transforming growth factor beta (TGFβ). Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a novel damage-associated molecular pattern (DAMP), is a TLR4 ligand and plays a role in a number of inflammatory disease processes. To test the hypothesis that eNAMPT is involved in NEC, an eNAMPT-neutralizing monoclonal antibody, ALT-100, was used in a well-established animal model of NEC. Preterm Sprague-Dawley pups delivered prematurely from timed-pregnant dams were exposed to hypoxia/hypothermia and randomized to control-foster mother dam-fed rats, injected IP with saline (vehicle) 48 h after delivery; control + mAB-foster dam-fed rats, injected IP with 10 µg of ALT-100 at 48 h post-delivery; NEC-orally gavaged, formula-fed rats injected with saline; and NEC + mAb-formula-fed rats, injected IP with 10 µg of ALT-100 at 48 h. The distal ileum was processed 96 h after C-section delivery for histological, biochemical, molecular, and RNA sequencing studies. Saline-treated NEC pups exhibited markedly increased fecal blood and histologic ileal damage compared to controls (q < 0.0001), and findings significantly reduced in ALT-100 mAb-treated NEC pups (q < 0.01). Real-time PCR in ileal tissues revealed increased NAMPT in NEC pups compared to pups that received the ALT-100 mAb (p < 0.01). Elevated serum levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), interleukin-8 (IL-8), and NAMPT were observed in NEC pups compared to NEC + mAb pups (p < 0.01). Finally, RNA-Seq confirmed dysregulated TGFβ and TLR4 signaling pathways in NEC pups that were attenuated by ALT-100 mAb treatment. These data strongly support the involvement of eNAMPT in NEC pathobiology and eNAMPT neutralization as a strategy to address the unmet need for NEC therapeutics.

Modification of Experimental Model of Necrotizing Enterocolitis (NEC) in Rat Pups by Single Exposure to Hypothermia and Hypoxia and Impact of Mother's Milk on Incidence of Disease.

BACKGROUND Necrotizing enterocolitis (NEC) is a potentially life-threatening disease that affects the intestine of the neonate, causing necrosis and general inflammation. Treatment consists of feeding cessation and antibiotic therapy. In more severe cases, surgical intervention is necessary. Recently, different NEC models have been used to study the development of novel diagnostic and therapeutic methods. This work modified an experimental NEC model in rat pups by a single exposure of animals to NEC-causing factors and testing the impact of mother's milk on prevalence of the disease. MATERIAL AND METHODS Fifty rat pups were subjected to the NEC protocol, in which they were exposed to 100% nitrogen atmosphere and cold stress for set periods of time and formula feeding with exposure to mother's milk and artificial milk. Twenty-nine pups were used for control. After a set time of 72 h, bowel fragments were obtained and examined histologically by hematoxylin-eosin staining with a modified 3-grade scale. RESULTS Histological features of NEC were present in most of the samples (10/14) in the group exposed to 1 min of hypoxia (P=0.016), 10 min of cold stress (P=0.4) and formula feeding every 3 h with no mother's milk (P=0.001). In the group of 11 animals with the same stress conditions but fed mother's milk right after birth, only 1 sample of NEC was present. CONCLUSIONS The modified experimental NEC model based on formula feeding and single exposure to hypothermia and hypoxia was assessed statistically and histologically. In this model, mother's milk had a protective effect against necrotizing enterocolitis.

Recombinant IGF-1/BP3 protects against intestinal injury in a neonatal mouse NEC model.

Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP3) is currently being tested in phase II clinical trials in premature infants to prevent bronchopulmonary dysplasia, but its impact on the neonatal intestine remains unclear. The aim of this study was to determine whether rhIGF-1/BP3 protects against necrotizing enterocolitis (NEC) in mice and to investigate the mechanisms involved. Neonatal mice were dam fed or injected intraperitoneally with rhIGF-1/BP3 (or vehicle) and submitted to an experimental NEC model. Serum IGF-1 was assessed by ELISA and intestinal vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression by Western blot. Intestinal endothelial cell proliferation, and enterocyte proliferation and migration were examined by immunofluorescence. Pup survival and histological intestinal injury were determined. In pups exposed to experimental NEC, serum IBP3-bound IGF-1 level was decreased. Exogenous rhIGF-1/BP3 preserved VEGF and VEGFR2 protein expression, decreased vascular permeability, and preserved endothelial cell proliferation in the small intestine. Furthermore, rhIGF-1/BP3 promoted enterocyte proliferation and migration, which effects were attenuated by inhibiting VEGFR2 signaling, decreased enterocyte apoptosis and decreased systemic and intestinal inflammation. rhIGF-1/BP3 improved survival and reduced the incidence of severe intestinal injury in experimental NEC. Exogenous rhIGF-1/BP3 protects neonatal mice against experimental NEC via multiple mechanisms. Exogenous rhIGF-1/BP3 preserves intestinal microvascular development and integrity, promotes enterocyte proliferation and migration, decreases local and systemic inflammation, and protects neonatal mice against NEC. The article adds pre-clinical evidence of a protective role for rhIGF-1/BP3 on the premature gut. It provides evidence supporting the use of rhIGF1/BP3 in premature neonates to protect against NEC.

Experimental necrotizing enterocolitis using oral lipopolysaccharide and protective role of breastmilk.

Necrotizing enterocolitis (NEC) is a life-threatening condition that afflicts neonates. Breastfeeding has demonstrated to play a protective role against it. By administering lipopolysaccharides (LPS) orally in newborn rats (NBR), we have developed an experimental model to induce NEC-like gut damage. Our aim was to assess the macroscopic and microscopic appearance of the gut, to evaluate the presence of NEC and study the role of breast milk (BM). NBR were divided into 3 groups: Group A (control, n=10) remained with the mother, group B (LPS, n=25) was isolated after birth, gavage-fed with special rat formula and oral LPS, then submitted to stress (hypoxia after gavage) and group c (BM, n=12) was breastfed once after birth, then isolated, and submitted to stress like group B. On day 4, NBR were sacrificed, and intestine was harvested and assessed. In the control group NEC was not present either macroscopically or histologically. Both groups submitted to stress (B and C) presented a global incidence of NEC of 73%. Most of group B developed histologic signs of NEC (85%) and group C showed a statistically lower incidence of NEC (50%, p=0.04), playing the BM a protective role against NEC (OR=0.19; 95% CI: 0.40-0.904). Our model showed a significant incidence of NEC in NBR (73%) with the same protective role of BM as in newborn humans, achieving a reliable and reproducible experimental NEC model. This will allow us to investigate new potential therapeutic targets for a devastating disease that currently lacks treatment.

Identification and evaluation of probiotic potential of Bifidobacterium breve AHC3 isolated from chicken intestines and its effect on necrotizing enterocolitis (NEC) in newborn SD rats.

Necrotizing enterocolitis (NEC) is a severe intestinal disease of the newborn infants, associated with high morbidity and mortality. It has been reported that Bifidobacterium could protect the intestinal barrier function and reduce the risk of NEC. This study aimed to evaluate the probiotic potential of Bifidobacterium strains isolated from the chicken intestines and its effect on necrotizing enterocolitis in newborn SD rats. Out of 32 isolates, B. breve AHC3 not only exhibited excellent probiotic potential, including tolerance to artificial simulated gastric conditions, adhesion to HT-29 cells, antioxidant capacity and antibacterial activity, but also possessed reliable safety. Additionally, NEC model was established to further investigate the effect of B. breve AHC3 on necrotizing enterocolitis in newborn SD rats. It was illustrated that administration of B. breve AHC3 significantly not only reduced the incidence of NEC (from 81.25% to 34.38%) (P< 0.05), but also alleviated the severity of ileal injury (P< 0.05). Compared with NEC model, B. breve AHC3 could significantly decrease the level of proinflammatory factor TNF-α (P< 0.05) and increase the level of antiinflammatory factor IL-10 (P< 0.05) in the ileum of NEC rats. Through the intervention of B. breve AHC3, the gray value of inducible nitric oxide synthase (iNOS) in intestinal tissue of NEC rats was significantly reduced (P< 0.05). It was indicated that B. breve AHC3 exhibited prominent probiotic potential and reliable safety. In the neonatal SD rat model of NEC, B. breve AHC3 had an available protective effect on the intestinal injury of NEC, which might be related to reducing the inflammatory reaction in the ileum and inhibiting the expression of iNOS in intestinal tissue cells. B. breve AHC3 could be used as a potential treatment for human NEC.

Effect of Oral Chondroitin Sulfate Supplementation on Acute Brain Injury in a Murine Necrotizing Enterocolitis Model.

Necrotizing enterocolitis (NEC) is a devastating condition where inflammatory changes and necrosis in the gut results in activation of brain microglia and subsequent neurodevelopmental impairment. Chondroitin sulfate (CS) is a glycosaminoglycan in human breast milk that is absent in conventional formulas. We hypothesized that oral formula supplementation with CS during a murine model of experimental NEC would not only attenuate intestinal injury, but also brain injury. NEC was induced in mouse pups on postnatal days (PNDs) 5 to 8. Three conditions were studied: (1) breastfed controls, (2) NEC, and (3) NEC+enteral CS (formula+200 mg/kg/d of CS). Pups were euthanized on PND 9 or reunited with dams by the evening of PND 8. Intestinal segments were H&E stained, and immunohistochemistry was performed on brain tissue for Iba-1 to assess for microglial morphology and cortical changes. Neurodevelopmental assays were performed on mice reunited with foster dams on PND 9. Single-cell RNA-sequencing analysis was performed on human intestinal epithelial cells exposed to (1) nothing, (2) hydrogen peroxide (H 2 O 2 ) alone, or (3) H 2 O 2 + CS to look at the differential gene expression between groups. Groups were compared with ANOVA or Kruskal-Wallis tests as appropriate with p < 0.05 considered significant. Compared with NEC, mice treated with oral CS showed improved clinical outcomes, decreased intestinal injury, and attenuated microglial activation and deleterious cortical change. Mice with CS performed better on early neurodevelopmental assays when compared with NEC alone. Single-cell analysis of HIEC-6 cells demonstrated that CS treatment down regulated several inflammatory pathways including nuclear factor κB-suggesting an explanation for the improved Th17 intestinal cytokine profile. Oral CS supplementation improved both physiological, clinical, and developmental outcomes. These data suggest that CS is a safe compound for formula supplementation for the prevention of NEC.

Superior performance of biofilm versus planktonic Limosilactobacillus reuteri in protection of the intestines and brain in a piglet model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related death in premature infants. Its etiology is multifactorial, with intestinal dysbiosis playing a major role. Probiotics are a logical preventative therapy for NEC, however their benefits have been inconsistent. We previously developed a novel probiotic delivery system in which planktonic (free-living) Limosilactobacillus reuteri (Lr) is incubated with biocompatible dextranomer microspheres (DM) loaded with maltose (Lr-DM-maltose) to induce biofilm formation. Here we have investigated the effects of Lr-DM-maltose in an enteral feed-only piglet model of NEC. We found a significant decrease in the incidence of Definitive NEC (D-NEC), death associated with D-NEC, and activated microglia in the brains of piglets treated with Lr-DM-maltose compared to non-treated piglets. Microbiome analyses using 16S rRNA sequencing of colonic contents revealed a significantly different microbial community composition between piglets treated with Lr-DM-maltose compared to non-treated piglets, with an increase in Lactobacillaceae and a decrease in Clostridiaceae in Lr-DM-maltose-treated piglets. Furthermore, there was a significant decrease in the incidence of D-NEC between piglets treated with Lr-DM-maltose compared to planktonic Lr. These findings validate our previous results in rodents, and support future clinical trials of Lr in its biofilm state for the prevention of NEC in premature neonates.

Interfering with Rac1-activation during neonatal monocyte-macrophage differentiation influences the inflammatory responses of M1 macrophages

Necrotizing enterocolitis (NEC) is a life-threatening, inflammatory disease affecting premature infants with intestinal necrosis, but the mechanism remains unclear. Neonatal macrophages are thought to play an important role in the pathogenesis of NEC through the production of proinflammatory cytokines. Restriction of cytokine expression in macrophages of NEC tissues may be beneficial. In adult macrophages, interfering with Rac1 has been shown to influence the expression of cytokines. Here, we investigated whether interfering with Rac1 in neonatal macrophages affects their inflammatory responses. First, we found that Rac1-activation was upregulated in the macrophages of rats with NEC model induction compared to controls. The M1 macrophages derived from human neonatal monocytes showed greater Rac1-activation than the M2 macrophages derived from the same monocytes. Inhibition of Rac1-activation by NSC23766 potently reduced the production of proinflammatory cytokines in these M1 macrophages. While neonatal monocytes differentiated into M1 macrophages in vitro, NSC23766 significantly altered cell function during the first six days of incubation with GM-CSF rather than during the subsequent stimulation phase. However, the same effect of NSC23766 was not observed in adult macrophages. Using mass spectrometry, Y-box binding protein 1 (YB1) was identified as being downregulated upon inhibition of Rac1-activation in the neonatal macrophages. Moreover, we found that inhibition of Rac1-activation shortens the poly A tail of PABPC1 mRNA, thereby reducing the translation of PABPC1 mRNA. Consequently, the downregulation of PABPC1 resulted in a reduced translation of YB1 mRNA. Furthermore, we found that TLR4 expression was downregulated in neonatal macrophages, while YB1 expression was reduced. Adding resatorvid (TLR4 signaling inhibitor) to the macrophages treated with NSC23766 did not further reduce the cytokine expression. These findings reveal a novel Rac1-mediated pathway to inhibit cytokine expression in neonatal M1 macrophages and suggest potential targets for the prevention or treatment of NEC.

Chondroitin sulfate supplementation improves clinical outcomes in a murine model of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) continues to be a devastating disease in preterm neonates and has a paucity of medical management options. Chondroitin sulfate (CS) is a naturally occurring glycosaminoglycan (GAG) in human breast milk (HM) and has been shown to reduce inflammation. We hypothesized that supplementation with CS in an experimental NEC model would alter microbial diversity, favorably alter the cytokine profile, and (like other sulfur compounds) improve outcomes in experimental NEC via the eNOS pathway. NEC was induced in 5-day-old pups. Six groups were studied (n = 9-15/group): (1) WT breastfed and (2) Formula fed controls, (3) WT NEC, (4) WT NEC + CS, (5) eNOS KO (knockout) NEC, and (6) eNOS KO NEC + CS. Pups were monitored for clinical sickness score and weights. On postnatal day 9, the pups were killed. Stool was collected from rectum and microbiome analysis was done with 16 s rRNA sequencing. Intestinal segments were examined histologically using a well-established injury scoring system and segments were homogenized and analyzed for cytokine profile. Data were analyzed using GraphPad Prism with p < 0.05 considered significant. CS supplementation in formula improved experimental NEC outcomes when compared to NEC alone. CS supplementation resulted in similar improvement in NEC in both the WT and eNOS KO mice. CS supplementation did not result in microbial changes when compared to NEC alone. Our data suggest that although CS supplementation improved outcomes in NEC, this protection is not conferred via the eNOS pathway or alteration of microbial diversity. CS therapy in NEC does improve the intestinal cytokine profile and further experiments will explore the mechanistic role of CS in altering immune pathways in this disease.

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

BackgroundHydrogen 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. MethodsNecrotizing 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. ResultsIn 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. ConclusionsGYY4137 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.

Effect of gut microbiota homeostasis on hematopoiesis in a neonatal rat model of necrotizing enterocolitis

To study the effect of gut microbiota on hematopoiesis in a neonatal rat model of necrotizing enterocolitis (NEC). Neonatal Sprague-Dawley rats were randomly divided into a control group and a model group (NEC group), with 6 rats in each group. Formula milk combined with hypoxia and cold stimulation was used to establish a neonatal rat model of NEC. Hematoxylin and eosin staining was used to observe the pathological changes of intestinal tissue and hematopoiesis-related organs. Routine blood tests were conducted for each group. Immunohistochemistry was used to observe the changes in specific cells in hematopoiesis-related organs. Flow cytometry was used to measure the changes in specific cells in bone marrow. 16S rDNA sequencing was used to observe the composition and abundance of gut microbiota. Compared with the control group, the NEC group had intestinal congestion and necrosis, damage, atrophy, and shedding of intestinal villi, and a significant increase in NEC histological score. Compared with the control group, the NEC group had significantly lower numbers of peripheral blood leukocytes and lymphocytes (P<0.05), nucleated cells in the spleen, thymus, and bone marrow, and small cell aggregates with basophilic nuclei in the liver (P<0.05). The NEC group had significant reductions in CD71+ erythroid progenitor cells in the liver, CD45+ lymphocytes in the spleen and bone marrow, CD3+ T lymphocytes in thymus, and the proportion of CD45+CD3-CD43+SSChi neutrophils in bone marrow (P<0.05). There was a significant difference in the composition of gut microbiota between the NEC and control groups, and the NEC group had a significant reduction in the abundance of Ligilactobacillus and a significant increase in the abundance of Escherichia-Shigella (P<0.05), which replaced Ligilactobacillus and became the dominant flora. Multi-lineage hematopoietic disorder may be observed in a neonatal rat model of NEC, which may be associated with gut microbiota dysbiosis and abnormal multiplication of the pathogenic bacteria Escherichia-Shigella.

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field's understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

The role of human milk nutrients in preventing necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an intestinal disease that primarily impacts preterm infants. The pathophysiology of NEC involves a complex interplay of factors that result in a deleterious immune response, injury to the intestinal mucosa, and in its most severe form, irreversible intestinal necrosis. Treatments for NEC remain limited, but one of the most effective preventative strategies for NEC is the provision of breast milk feeds. In this review, we discuss mechanisms by which bioactive nutrients in breast milk impact neonatal intestinal physiology and the development of NEC. We also review experimental models of NEC that have been used to study the role of breast milk components in disease pathophysiology. These models are necessary to accelerate mechanistic research and improve outcomes for neonates with NEC.

Intestine and brain TLR-4 modulation following N-acetyl-cysteine treatment in NEC rodent model

Necrotizing enterocolitis (NEC) brain injury is mediated through Toll-like receptor 4 (TLR4) on the intestinal epithelium and brain microglia. Our aim was to determine whether postnatal and/or prenatal NAC can modify NEC associated intestinal and brain TLR4 expression and brain glutathione levels in a rat model of NEC. Newborn Sprague–Dawley rats were randomized into three groups: Control (n = 33); NEC (n = 32)—hypoxia and formula feeding; and NEC-NAC (n = 34)—received NAC (300 mg/kg IP) in addition to NEC conditions. Two additional groups included pups of dams treated once daily with NAC (300 mg/kg IV) for the last 3 days of pregnancy: NAC-NEC (n = 33) or NAC-NEC-NAC (n = 36) with additional postnatal NAC. Pups were sacrificed on the fifth day, and ileum and brains harvested for TLR-4 and glutathione protein levels. Brain and ileum TLR-4 protein levels were significantly increased in NEC offspring as compared to control (brain 2.5 ± 0.6 vs. 0.88 ± 0.12 U and ileum 0.24 ± 0.04 vs. 0.09 ± 0.01, p < 0.05). When NAC was administered only to dams (NAC-NEC) a significant decrease in TLR-4 levels was demonstrated in both offspring brain (1.53 ± 0.41 vs. 2.5 ± 0.6 U, p < 0.05) and ileum (0.12 ± 0.03 vs. 0.24 ± 0.04 U, p < 0.05) as compared to NEC. The same pattern was demonstrated when NAC was administered only or postnatally. The decrease in brain and ileum glutathione levels observed in NEC offspring was reversed with all NAC treatment groups. NAC reverses the increase in ileum and brain TLR-4 levels and the decrease in brain and ileum glutathione levels associated with NEC in a rat model, and thus may protect from NEC associated brain injury.

Microfluidic device facilitates in vitro modeling of human neonatal necrotizing enterocolitis-on-a-chip.

Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants that is associated with an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. We describe an in vitro model of human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology. This model utilizes premature infant intestinal enteroids grown from surgically harvested intestinal tissue and co-cultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology by adding infant-derived microbiota. This model, named NEC-on-a-Chip, recapitulates the predominant features of NEC including significant upregulation of pro-inflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation, and disrupted epithelial barrier integrity. NEC-on-a-Chip provides an improved preclinical model of NEC that facilitates comprehensive analysis of the pathophysiology of NEC using precious clinical samples. This model is an advance towards a personalized medicine approach to test new therapeutics for this devastating disease.

Development of a novel definitive scoring system for an enteral feed-only model of necrotizing enterocolitis in piglets.

Necrotizing enterocolitis (NEC) is a complex inflammatory disorder of the human intestine that most often occurs in premature newborns. Animal models of NEC typically use mice or rats; however, pigs have emerged as a viable alternative given their similar size, intestinal development, and physiology compared to humans. While most piglet NEC models initially administer total parenteral nutrition prior to enteral feeds, here we describe an enteral-feed only piglet model of NEC that recapitulates the microbiome abnormalities present in neonates that develop NEC and introduce a novel multifactorial definitive NEC (D-NEC) scoring system to assess disease severity. Premature piglets were delivered via Caesarean section. Piglets in the colostrum-fed group received bovine colostrum feeds only throughout the experiment. Piglets in the formula-fed group received colostrum for the first 24 h of life, followed by Neocate Junior to induce intestinal injury. The presence of at least 3 of the following 4 criteria were required to diagnose D-NEC: (1) gross injury score ≥4 of 6; (2) histologic injury score ≥3 of 5; (3) a newly developed clinical sickness score ≥5 of 8 within the last 12 h of life; and (4) bacterial translocation to ≥2 internal organs. Quantitative reverse transcription polymerase chain reaction was performed to confirm intestinal inflammation in the small intestine and colon. 16S rRNA sequencing was performed to evaluate the intestinal microbiome. Compared to the colostrum-fed group, the formula-fed group had lower survival, higher clinical sickness scores, and more severe gross and histologic intestinal injury. There was significantly increased bacterial translocation, D-NEC, and expression of IL-1α and IL-10 in the colon of formula-fed compared to colostrum-fed piglets. Intestinal microbiome analysis of piglets with D-NEC demonstrated lower microbial diversity and increased Gammaproteobacteria and Enterobacteriaceae. We have developed a clinical sickness score and a new multifactorial D-NEC scoring system to accurately evaluate an enteral feed-only piglet model of NEC. Piglets with D-NEC had microbiome changes consistent with those seen in preterm infants with NEC. This model can be used to test future novel therapies to treat and prevent this devastating disease.

State-of-the-art review and update of in vivo models of necrotizing enterocolitis.

NEC remains one of the most common causes of mortality and morbidity in preterm infants. Animal models of necrotizing enterocolitis (NEC) have been crucial in improving our understanding of this devastating disease and identifying biochemical pathways with therapeutic potential. The pathogenesis of NEC remains incompletely understood, with no specific entity that unifies all infants that develop NEC. Therefore, investigators rely on animal models to manipulate variables and provide a means to test interventions, making them valuable tools to enhance our understanding and prevent and treat NEC. The advancements in molecular analytic tools, genetic manipulation, and imaging modalities and the emergence of scientific collaborations have given rise to unique perspectives and disease correlates, creating novel pathways of investigation. A critical review and understanding of the current phenotypic considerations of the highly relevant animal models of NEC are crucial to developing novel therapeutic and preventative strategies for NEC.