Pediatr Surg Int DOI 10.1007/s00383-015-3697-9

REVIEW ARTICLE

Pathogenesis of neonatal necrotizing enterocolitis Joanna C. Lim1 • Jamie M. Golden1 • Henri R. Ford1,2

Accepted: 23 March 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Although necrotizing enterocolitis (NEC) is the most lethal gastrointestinal disease in the neonatal population, its pathogenesis is poorly understood. Risk factors include prematurity, bacterial colonization, and formula feeding. This review examines how mucosal injury permits opportunistic pathogens to breach the gut barrier and incite an inflammatory response that leads to sustained overproduction of mediators such as nitric oxide and its potent adduct, peroxynitrite. These mediators not only exacerbate the initial mucosal injury, but they also suppress the intestinal repair mechanisms, which further compromises the gut barrier and culminates in bacterial translocation, sepsis, and full-blown NEC. Keywords Necrotizing enterocolitis  Pediatric surgery  Neonatal intensive care unit  Pathophysiology

Introduction Necrotizing enterocolitis (NEC) is the most common and most lethal disorder affecting the gastrointestinal tract of newborn infants. The incidence of NEC is approximately 1.1 per 1000 live births in the general population, but it affects up to 7 % of preterm infants with birth weight between 500 and 1500 g [1]. Recent advances in neonatology

& Henri R. Ford [email protected] 1

Division of Pediatric Surgery, Children’s Hospital Los Angeles, 4650 Sunset Blvd., Mailstop #72, Los Angeles, CA 90027, USA

2

Department of Surgery, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA

have resulted in not only improved survival rates in this group but also, in a concomitant increase in the incidence of NEC. Overall NEC-associated mortality is estimated to be 20–30 % [2]. Despite improvements in the medical management of NEC, 20–40 % of affected patients will require surgical intervention, and in that population, morbidity and mortality approach 20–50 % [3]. Approximately 25 % of NEC survivors suffer from serious comorbidities including short gut syndrome and neurodevelopmental impairment. Thus, NEC remains one of the most challenging problems affecting the premature infant. NEC is a severe inflammation of the small intestine that is characterized by feeding intolerance, abdominal distention, bloody stool, and pneumatosis intestinalis. Advanced disease may be associated with thrombocytopenia, systemic sepsis, and pneumoperitoneum. Histologically, there are varying degrees of epithelial sloughing, submucosal edema, neutrophil infiltration, and destruction of villus architecture. Initial management of NEC consists of bowel rest, fluid resuscitation, and broad-spectrum antibiotics. These treatments may or may not provide relief. If the disease progresses, surgical resection of the affected tissue may become necessary. This is often associated with short gut syndrome and its accompanying adverse sequelae. NEC should be diagnosed and treated early to avoid its devastating consequences. Unfortunately, there are neither reliable tools to predict nor effective strategies to prevent and treat NEC. This problem stems from our poor understanding of the pathogenesis of this devastating disease. Although the exact etiology of NEC is not well understood, known risk factors include prematurity, formula feeding, and bacterial colonization of the gut. The intestine of a preterm neonate is characterized by underdeveloped immune defenses and compromised mucosal barrier function [4]. As a result, the immature intestine is susceptible to

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bacterial colonization by opportunistic pathogens, which often follows oral feeding. Premature infants experience hypoxia and hypothermia, which may predispose them to intestinal mucosal injury. These factors set the stage for intestinal barrier breakdown and invasion by opportunistic pathogens, which incite an exuberant inflammatory response culminating in the adverse sequelae of NEC.

Environmental factors Bacteria The key role of bacteria in the pathogenesis of NEC has helped in classifying NEC as an infectious disease. In a rodent model of NEC, germ-free mice failed to develop NEC [5]. However, beyond this key finding, the interplay between bacteria and neonatal human host is still incompletely understood, and the precise bacterial triggers for NEC have yet to be found. Evidence suggests that the neonatal intestinal microbiota plays a key role in health and disease, with certain profiles correlating with childhood atopy and obesity [6]. However, because the colonization process is so capricious and fluid, the question of what constitutes normal versus abnormal microbiota in neonates is still unresolved [7]. The composition of the human microbiota is in constant flux during the first 2 years of life, after which it stabilizes and resembles adult flora. It is known that intestinal microbiota is acquired from the environment. This is supported by the finding that the gastrointestinal tract of babies born by Cesarean section is first colonized by skin flora, while that of babies delivered vaginally is first colonized by adult gastrointestinal flora [8, 9]. Early microbiota have few characteristic profiles; however, there is a trend toward low diversity and high inter-individual variability of predominant colonizing species [10]. When these studies were repeated in the preterm population, these trends became even more noticeable [11, 12]. One distinct pattern is that breast-fed infants have a predominance of Bifidobacteria species compared with formula-fed infants, who have more coliforms, enterococci, and bacteroides [13, 14]. This trend still persists at 1 month of age when all infants show a prevalence of Bifidobacteria; however, breast-fed infants show a tenfold greater quantity of these organisms compared with formula-fed ones [15]. Another distinctive trait is that infants requiring prolonged hospitalization have decreased exposure to environmental flora and are subject to increased antibiotic administration, which further delays establishment of the normal intestinal microflora [13, 16– 18]. This delay in intestinal colonization potentially predisposes to NEC because protective bacterial species may

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not be in place to counterbalance or limit colonization by opportunistic pathogens. Efforts to identify bacterial species and patterns of microbiota associated with NEC have thus far had limited success [19–21]. Bacterial species found in NEC patients have also been found in healthy individuals, and there have been no attempts to establish a causal relationship between specific bacteria and the disease. Yet despite the limitations of microbiota studies, extensive data on hospital outbreaks in which clustered cases of NEC were associated with specific strains of bacteria, viruses, or fungi clearly point to the infectious nature of this disease [22]. The contagious nature of NEC was confirmed through a study in gnotobiotic quails, where Clostridium species obtained from the stool of human infants affected with NEC caused similar cecal lesions, including intramural gaseous cysts, hemorrhagic ulcerations, and intestinal necrosis [23]. Conversely, whole fecal flora from a healthy infant, which naturally included Bifidobacterium but not Clostridium, did not produce NEC-like lesions. Cronobacter muytjensii, previously known as Enterobacter sakazakii, is one of the bacterial species implicated as the causative agent in several hospital outbreaks of NEC. Hunter et al. established that C. muytjensii dramatically increases the incidence and severity of NEC in neonatal rats [24, 25]. Moreover, the ability of C. muytjensii to promote NEC was not a property of the bacterial species as a whole but rather a characteristic of certain strains, which might explain why bacteria of the same species could be pathogenic or non-pathogenic [26]. The pathogenic C. muytjensii also caused enterocyte apoptosis and increased the production of interleukin-6 [24]. Preinoculation with Lactobacillus bulgaricus abrogated C. muytjensii-mediated intestinal inflammation both in vivo and in vitro [25]. Although these findings clearly implicate bacteria in the development of NEC, the details regarding pathogenesis are still under investigation. There has been a growing interest in targeting the abnormal microbial flora in the treatment and prevention of NEC [27]. From clearing ‘‘bad’’ bacteria with antibiotics to augmenting ‘‘good’’ bacteria with probiotics or fecal microbiota transplantation (FMT), there is much needed to study in the field of NEC microbiota. Current standard of care dictates prompt administration of broad-spectrum antibiotics at initial suspicion of NEC to eradicate the putative opportunistic pathogen(s). However, because most preterm infants receive antibiotics for various reasons, the efficacy of this clinical practice is difficult to ascertain. Prophylaxis with oral antibiotics has been shown to reduce the incidence of NEC, but it also caused colonization with antibiotic-resistant bacteria and thus has

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not been widely accepted as standard practice [28]. For preterm low-birth weight infants, the American Pediatric Surgical Association recommends prophylactic probiotic or human milk administration over formula [29]. The replacement of pathogenic bacteria with protective bacteria has led to a growing interest in probiotics and FMT to combat NEC. Probiotics have been shown to enhance host defense mechanisms and the humoral immune response [30]. Chen and Walker outlined potential mechanisms of probiotic effects in NEC [31]: 1. 2. 3. 4. 5.

Reduced mucosal colonization by potential pathogens. Increased barrier to translocation of bacteria and bacterial products across the intestinal mucosa. Competitive exclusion of potential pathogens from enterocyte interaction. Activation of the host’s mucosal response to microbial products. General upregulation of intestinal protective function.

Clinically, routine administration of probiotics such as Lactobacillus sp. or Bifidobacteria sp. reduced the incidence of NEC in multiple randomized controlled trials [32– 35]. However, this practice has yet to be widely accepted because probiotics do not have a clear mechanism of action, have not been studied in extremely low-birth weight infants who are at the greatest risk of side effects, and have not been evaluated for long-term impact [36, 37]. Enteral nutrition Preterm infants receiving breast milk have lower incidence of NEC compared with their formula-fed counterparts [38, 39]. As previously mentioned, breast milk shifts the intestinal microbiota to a more favorable profile by promoting protective strains such as Bifidobacteria and decreasing potential pathogenic strains such as coliforms. The immunomodulatory effects of breast milk also add to the superiority of breast-feeding. Breast milk confers protection through a number of factors including secretory IgA, antimicrobial proteins such as CD14, cytokines, epidermal growth factor, and fatty acids, to name a few [40]. Several studies have shown the positive effect of breast milk. Oral gavage of human milk oligosaccharide disialyllacto-N-tetraose decreased the incidence of NEC [41]. In another study, breast-fed neonatal rodents showed increased expression of P-glycoprotein (Pgp) compared with formula-fed rodents. Silencing the Pgp-producing MDR1a gene resulted in a higher incidence of NEC [42]. Several studies have examined the efficacy of delayed enteral feeding in reducing the incidence of NEC. However, none of these studies or meta-analyses showed any advantage to delayed feeding strategies [43, 44].

Immature intestine The neonatal intestine is inherently fragile with increased permeability and an immature immune system, making it vulnerable to environmental threats or internal stressors such as hypoxia or ischemia. Multiple growth factors act on the developing intestine to enhance gut barrier function. Growth factors NEC is characterized by breakdown of the intestinal epithelial lining. One explanation for the disease may be an inability to prevent or recover from mucosal injury, which has prompted investigation into growth factors that mediate intestinal development and maturation. Epidermal growth factor (EGF) is a trophic factor in intestinal development, critical for epithelial cell proliferation and survival [45, 46]. The amniotic fluid concentration of EGF increases with gestational age [47]. Initially after birth, intestinal EGF is derived primarily from maternal colostrum and breast milk until the salivary glands develop and take over production [48–50]. In utero EGF exposure in a rabbit model showed increased intestinal growth and accelerated maturation of intestinal enzyme activity [51]. Besides its role in initial intestinal development, EGF has also been shown to aid in recovery from injury or disease. In mucosal ulcer disease, the absence of EGF has been associated with disease progression, while EGF supplementation aided repair and regeneration [45]. Following massive small bowel resection, EGF is increased and necessary for adaptation of the remaining intestine [52–60]. Therefore, it is logical that NEC may be countered by EGF. EGF receptor (EGFR) mRNA expression was increased when intestinal repair was underway in experimental NEC [61, 62]. EGF prevented NEC by increasing and stimulating mucin-secreting goblet cells and restoring tight junction protein expression in an experimental model of NEC [63]. Heparin-binding epidermal-like growth factor (HBEGF) is a member of the EGF family that binds strongly to heparin. HB-EGF is also found in amniotic fluid and in human milk, and its expression is elevated in response to injury, such as tissue damage, hypoxia, and stress [64]. In vitro and in vivo studies of intestinal anoxia/reoxygenation and ischemia/reperfusion show that HB-EGF increases after injury and improves epithelial restitution by acting on EGFR [65]. Intestinal segments resected from infants with NEC showed increased HB-EGF mRNA expression in the healthy tissue margin compared with the frankly necrotic tissue, suggesting that the relative increase contributes to healing or, conversely that the relative

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deficiency contributes to NEC. HB-EGF supplementation in a neonatal rat model decreased NEC incidence, reduced intestinal apoptosis, and enhanced enterocyte migration and proliferation [66–68]. Other growth factors have been identified in the development and maintenance of the neonatal intestine including glucagon-like peptide 2 (GLP-2), growth hormone (GH), insulin-like growth factor-1 (IGF-1), granulocyte colonystimulating factor (G-CSF), erythropoietin (Epo), intestinal trefoil factor (ITF), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) [45]. Each of these growth factors constitutes a potential therapeutic target in the prevention and treatment of NEC. Microcirculation In the neonatal intestine, blood flow in the fragile microcirculation is mainly determined by resting vascular resistance [69]. Extrinsically, this is affected by the autonomic nervous system and cardiovascular reflexes. Intrinsically, the microcirculation is managed by local mediators from the intestine and its vasculature. Newborns have a lower resting vascular resistance in the intestinal microcirculation, thus having a relative increase in blood flow. Two factors that mediate this delicate balance are the vasoconstrictor—endothelin-1 (ET-1), and the vasodilator—nitric oxide (NO). Endothelin-1 is the primary vasoconstrictor of the neonatal intestinal microcirculation. Produced by endothelial cells, this protein acts by binding to endothelin receptor type A (ETA) and endothelin receptor type B (ETB). ET-1 is constitutively expressed, and under basal conditions, ET-1 production is greater in younger subjects compared with older ones [70]. Both endothelin receptors, ETA and ETB, are expressed to a greater extent in younger patients relative to older ones. ETA receptors are located on vascular smooth muscle cells, and their activation induces vasoconstriction. ETB receptors are expressed on vascular smooth muscle cells and endothelial cells, and their activation induces NO-mediated vasodilation. In the neonatal intestinal microcirculation, ETA vasoconstriction outweighs ETB vasodilation; thus, the net effect of endothelin1 is vasoconstriction. Ischemia-mediated intestinal mucosal injury is believed to play an important role in NEC pathogenesis. When ET-1 was examined in a rat model of NEC, the ileum showed decreased intestinal blood flow and increased ET-1 mRNA expression [71]. Topical application of ET-1 to the intestinal mucosa in this model exacerbated the hypoperfusion. These findings were corroborated by human studies, which showed that increased ET-1 expression in NEC specimens correlated with the degree of intestinal injury [72].

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NO is a free radical that modulates many physiologic processes including vascular tone, tissue homeostasis, neurotransmission, platelet aggregation, mucosal integrity, reperfusion injury, and inflammation [73–76]. It acts locally via paracrine effects due to its short half-life, solubility in water and lipids, permeability, and ability to diffuse rapidly [77]. It forms when NO synthase (NOS) reacts with arginine and oxygen to form citrulline and NO. There are three isoforms of NOS: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). eNOS and nNOS are constitutive and produce low levels of NO, while iNOS is induced by inflammatory stimuli to produce high levels of NO. All three isoforms are found in the intestine [78]. NO plays a paradoxical role in intestinal cell homeostasis. NO produced constitutively helps maintain mucosal blood flow and inhibits platelet aggregation and adhesion of leukocytes [75, 79]. It also plays a role in host defense against microbes due to its antimicrobial properties [80]. However, high levels of NO produced by iNOS can be toxic, and its sustained upregulation under inflammatory conditions can exert cytopathic effects on the intestinal epithelium. NO can react either with oxidants to form stable nitrogen oxides (such as nitrite or nitrate) or with other free radicals to produce more toxic adducts that may cause tissue injury [77]. High levels of NO cannot be rapidly scavenged by red blood cells to form stable compounds; therefore, NO reacts with superoxide to produce peroxynitrite. Peroxynitrite is a highly toxic and potent intermediate that directly oxidizes target proteins, forms highly reactive radicals, and can modify tyrosine residues forming nitrotyrosine. Nitrotyrosine interferes with enzymatic activity and intracellular signaling [81]. This free radicalinduced damage plays a key role in inflammatory gut barrier failure. High levels of iNOS have been implicated in intestinal barrier breakdown in both human and experimental NEC. High levels of NO and peroxynitrite both enhance intestinal injury in NEC by promoting apoptosis and impairing intestinal restitution by decreasing enterocyte proliferation and migration [82–85]. High levels of iNOS mRNA and peroxynitrite were found in intestine resected from infants with acute NEC, and these levels returned to baseline at the time of stoma closure in these infants [83]. Similar findings were reported in a neonatal rat model of NEC [86]. In addition, arginine, the precursor of NO, was decreased in the plasma of infants with NEC [87]. The role of iNOS in gut barrier dysfunction was also investigated in endotoxin or lipopolysaccharide (LPS)-induced peritonitis. LPS administration resulted in iNOS upregulation and gut barrier failure. Intestinal permeability in this model was decreased with iNOS inhibitors [88]. Furthermore, mice either

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deficient in iNOS or treated with a NO inhibitor (aminoguanidine) were protected from gut barrier failure in experimental peritonitis [84, 89–91]. Thus, sustained overproduction of NO and its adduct, peroxynitrite, lead to gut barrier failure not only by causing mucosal injury, but also by impairing mucosal healing, which culminates in bacterial translocation, sepsis, and NEC [78].

and postnatal mouse ileum, the gut had higher expression of TLR4 [100]. Hypoxia and LPS exposure were also found to increase TLR4 expression, thus potentially contributing to NEC. In term babies, TLR4 levels decrease shortly after birth, which allows for intestinal mucosal adaptation to commensal flora. In preterm babies, TLR4 levels remain high, consistent with its role in intestinal development, exaggerating TLR4-mediated cytokine response and predisposing the gut to barrier breakdown and NEC.

Inflammatory cascade Platelet-activating factor Multiple pro-inflammatory mediators have been associated with NEC. The immature intestine is capable of mounting an exaggerated inflammatory response. Indeed, fetal tissue shows increased expressions of TLR2, TLR4, MyD88, TRAF-6, NFkB1, and IL-8 mRNA and decreased expressions of SIGIRR, IRAK-M, A-20, and TOLLIP mRNA compared with intestine from older children [92]. Intestine from NEC patients had similar changes in inflammatory mRNA levels but to a greater degree than mature intestine from older children. TLR4 signaling Toll-like receptor 4 (TLR4) is expressed on leukocytes and serves as the receptor for LPS. Upon LPS exposure, TLR4 initiates the inflammatory cascade by activating the transcription factor NF-jB. The LPS-TLR4 interaction is also crucial for phagocytosis of bacteria [93]. TLR4 is critical for in utero intestinal differentiation and innate immunity [94]. However, although TLR4 may play a role in natural defense against Gram-negative bacteria, current research suggests that its overexpression in premature infants and its destructive downstream effects can lead to NEC [94, 95]. In addition to leukocytes, TLR4 is also expressed in the intestinal epithelium and negatively regulates enterocyte migration and proliferation after injury [96]. Inhibition of migration is due to increased adhesiveness mediated by expression of matrix-binding molecules known as integrins and focal adhesion kinase. TLR4 also inhibits b-catenin signaling, interfering with the degree of enterocyte proliferation in the small intestine [97]. This suppression of intestinal repair mechanisms contributes to gut barrier failure. TLR4 gene deletion resulted in normal enterocyte proliferation and reduced apoptosis in knockout mice compared with their wild-type counterparts [98]. Similarly, TLR4 mutants developed less NEC than their wild-type counterparts, and histological NEC specimens showed elevated TLR4 mRNA expression [99]. TLR4-mediated decrease in enterocyte proliferation in wild types resulted in greater intestinal breakdown, consistent with NEC. Intestinal expression of TLR4 may also explain why premature infants are more susceptible to NEC. In prenatal

Platelet-activating factor (PAF) is a potent phospholipid inflammatory mediator, activation of which results in apoptosis, epithelial cell damage, increased mucosal permeability, impaired tight junctions, leukocyte and platelet aggregation, and vasoconstriction [101]. PAF-specific phospholipase A2 is the rate-limiting step in the synthesis of PAF, and its degradation is mediated by PAF-acetylhydrolase (PAF-AH). PAF-AH presence is low at birth and slowly increases to adult levels by 6 weeks of life [102]. Direct vascular infusion of PAF with LPS in adult rats caused ischemic bowel necrosis, histological findings that resemble NEC [103]. Pretreatment with dexamethasone and medroxyprogesterone resulted in increased PAF-AH and prevented gut barrier breakdown [104]. Intraluminal infusion of PAF in adult rat ileum caused intestinal damage and also resulted in TLR4 upregulation [105]. In neonatal rat models of NEC, the PAF receptor antagonist WEB 2170 decreased the incidence of NEC [106]. Similarly, enteral PAF-AH supplementation decreased the incidence of NEC [107]. Investigators have reported elevated levels of PAF and lower levels of PAF-AH in human NEC compared with controls [108]. Elevated stool PAF concentrations were detected (1) when enteral nutrition was initiated in preterm neonates, and (2) in patients with NEC [108, 109]. Although neonates are initially deficient in PAF-AH, maternal breast milk is an exogenous source that can compensate for its relative absence in the first few weeks of life [110, 111]. IL-8 Interleukin 8 (IL-8) is an inflammatory cytokine that can be secreted by any cell with Toll-like receptors, including epithelial cells and macrophages. Also known as neutrophil chemotactic factor, IL-8 draws neutrophils toward sites of infection and then induces phagocytosis. When LPS was given to human fetal and child small intestine organ cultures, the IL-8 secretion was greater in the fetal tissue, supporting the idea of the premature intestine as a hyperresponsive inflammatory setting [112].

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COX-2 Cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) are inflammatory mediators found in the intestine that play a key role in inflammatory bowel disease and NEC. Prostanoids are lipids derived from membrane arachidonic acid via cyclooxygenation and peroxidation. Cyclooxygenase is the rate-limiting step in prostanoid biosynthesis and converts arachidonic acid into prostaglandin H2 (PGH2). PGH2 is then converted into prostaglandin D2, E2, F2, and I2 by cell-specific prostaglandin synthases [113–115]. Prostanoids regulate blood flow, apoptosis, proliferation, migration, angiogenesis, gastrointestinal secretions, smooth muscle tone, body temperature, pain sensation, and inflammation [73, 116–120]. PGE2 is the major prostanoid in the intestine, and it acts on 4 transmembrane G protein-coupled receptors, EP1-4. EP1 activates phospholipase C (PLC) resulting in downstream activations of protein kinase C (PKC) and intracellular release of calcium. EP2 and EP4 activate adenylyl cyclase (AC), which leads to downstream activation of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA). EP3 downregulates AC. The various effects caused by PGE2 are attributed to these four different receptors and their differential expression in the GI tract [113, 115]. Similar to NO, cyclooxygenase and prostaglandin E2 play a paradoxical role in intestinal homeostasis. There are three isoforms of cyclooxygenase: COX-1, COX-2, and COX-3. COX-1 and COX-3 are expressed constitutively, while COX-2 is inducible. Cyclooxygenase activity is essential as demonstrated by the early post-partum death of COX-1 and COX-2 double knockout mice [121]. Low levels of COX-2 and PGE2 have protective effects and are critical for maintaining the intestinal epithelium, while high levels that are induced during inflammation can be detrimental. Increased expression of COX-2 seen in inflammatory bowel disease and NEC is implicated in increased gut barrier permeability and bacterial translocation [113, 121–123]. Elevated levels of COX-2 have been reported in animal models of NEC as well as human tissue samples from infants with NEC [124–126]. Paradoxically, inhibition of COX-2 can also exacerbate inflammation and worsen the disease process. Systemic administration of indomethacin, a non-selective COX inhibitor commonly used in premature infants to close a patent ductus arteriosus, has been associated with the development of NEC [127]. Furthermore, administration of PGE2 increases intestinal blood flow in a neonatal rat model of NEC, thus supporting the protective effects of low levels of COX-2 and PGE2 [128]. Administration of a COX-2 inhibitor, Celecoxib, in a neonatal rat model of NEC worsened disease severity

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[125]. However, low doses of Celecoxib, but not high doses, were protective against gut barrier failure in experimental peritonitis. Low-dose Celecoxib may decrease levels of COX-2 without eliminating its protective effects [122]. The effects of COX-2 and PGE2 in intestinal barrier homeostasis are complex and concentration dependent. Potential COX-2-related therapy must target high levels of COX-2 that can lead to intestinal barrier breakdown, while avoiding complete elimination of COX-2 activity in order to preserve its protective effects on the intestinal mucosa.

Conclusion In conclusion, NEC is a disease etiology of which is still unclear, in large part because it occurs in the shifting environment of the neonatal intestine where many processes are evolving concurrently. A key contributing factor is early intestinal colonization by opportunistic pathogens rather than protective bacteria, which may be partly related to antibiotic therapy in the NICU. In addition, in the premature infant, the epithelial lining, vascular supply, and immune defenses are still developing. Disruption of any of these processes, especially following a hypoxic or ischemic insult, may result in mucosal barrier breakdown, bacterial invasion, and activation of the inflammatory cascade leading to a downward spiral characterized by uncontrolled inflammation, tissue destruction, systemic sepsis, and all of the adverse sequelae of NEC. As we continue to elucidate the normal development of the intestinal microbiota, gut barrier, and immune function, we will achieve a deeper understanding of how the complex interplay among these components leads to the development of NEC.

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Pathogenesis of neonatal necrotizing enterocolitis.

Although necrotizing enterocolitis (NEC) is the most lethal gastrointestinal disease in the neonatal population, its pathogenesis is poorly understood...
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