Necrotizing enterocolitis (NEC) remains one of the most commonly acquired gastrointestinal (GI) and surgical emergencies in preterm very-low-birth-weight (PVLBW) infants. In fact, its incidence and associated deaths are on the rise (1). Recent reports show that the incidence of NEC is inversely proportional to birth weight, with the illness affecting 11.5% of infants weighing between 401 and 750 g, 9% of those weighing between 751 and 1000 g, 6% of those weighing between 1001 and 1250 g, and 4% of those weighing between 1251 and 1500 g. Mortality ranges from 10% to 50%.
NEC often rapidly progresses from early signs of intestinal inflammation to extensive necrosis within a matter of hours, making secondary prevention equally if not more difficult to achieve. Thus, primary prevention should be the priority; however, despite >3 decades of exertion, little progress has been made in the prevention of NEC. Primary prevention remains elusive because of the complex pathogenesis. Fortunately, recent studies provide information that enhances our understanding of the pathophysiology and provides more practical options for prevention of NEC. This review focused on the present understanding of the pathophysiology of NEC in the context of developing optimal preventive strategies for PVLBW infants.
The pathogenesis of NEC is multifactorial (Fig. 1); however, the immaturity of the GI tract in terms of motility, digestive function, circulatory regulation, barrier function, and immune defense is the single most important underlying risk factor. The widely accepted hypothesis at present is that enteral feeding (providing substrate) in the presence of intestinal colonization by pathogens provokes an inappropriately accentuated inflammatory response by immature intestinal epithelial cells of preterm neonates (2).
Given that NEC occurs much later (median age of onset 21 days in neonates younger than 28 weeks at birth) in preterm neonates (3), perinatal hypoxic-ischemic events are less plausible in the pathogenesis of preterm NEC; researchers have questioned the ability of asphyxia models to accurately reflect the pathological changes in preterm NEC (4). Furthermore, most neonates with hypoxic-ischemic injury do not develop NEC (5), and no inciting hypoxic-ischemic event is identifiable in many neonates with NEC.
IMMATURITY OF INTESTINAL FUNCTIONS
Many prematurity-related limitations of intestinal functions may contribute to the development of NEC in preterm neonates. First, there is the reduced integrity of epithelial tight junctions. Then there is the impaired peristalsis and deficiencies in components of the mucus lining (6). There is also cow's-milk protein (7) and carbohydrate maldigestion (8). Tight junctions between epithelial cells of the intestinal tract serve as a protective barrier. Any breach or disruption of this barrier can activate the inflammatory response from the sensitive submucosal tissue. Release of proinflammatory cytokines after reperfusion injury further disrupts the integrity of the gut barrier and results in intestinal hyperpermeability and initiates a vicious cycle that involves worsening intestinal injury and inflammation that characterize the clinical picture of NEC.
Intestinal epithelial integrity is regulated by cyclooxygenases (COXs), nitric oxide (NO), and endothelium growth factor (EGF). COXs are critical for maintaining the intestinal epithelium. Studies suggest a paradoxical role of COX-2: beneficial under normal circumstances but deleterious under inflammatory conditions such as NEC (9). Mishima et al (10) reported that iNOS knockout mice were protected from gut barrier failure and other deleterious effects of endotoxemia. NO also plays a paradoxical role in intestinal physiology: low levels enhance mucosal blood flow and are important in maintaining mucosal integrity, but sustained elevated levels become cytotoxic to the gut epithelium (11). The damage involves direct epithelial and mitochondrial injury through membrane oxidation and induction of epithelial apoptosis.
Serum and salivary levels of EGF are decreased in preterm neonates with NEC such that salivary EGF levels in the first 2 weeks of life may be predictive of NEC in preterm neonates. In animal models of NEC, EGF administration increases intestinal barrier strength and reduces disease severity (12).
Clinical and experimental studies have shown that peristalsis of the GI tract is developmentally regulated and impaired in preterm infants until approximately 32 weeks of gestation; however, after birth, external factors (eg, feeding) augment the maturation of peristalsis in preterm neonates. Fetal or perinatal hypoxia further decreases intestinal motility (1). Impaired peristalsis not only allows for increased carbohydrate load from enteral feeding to serving as bacterial substrate but also prolongs gut exposure to bacterial antigens, thereby inducing an inflammatory response.
Immunoglobulin A (IgA), which is present in mucous secretions, provides protection against pathogenic organisms. Deficient IgA production in preterm neonates may facilitate bacterial translocation across the intestinal mucosa. One explanation for the reduced incidence of NEC in infants fed with human milk is the enhanced production of IgA in human milk feeding (13).
Preterm neonates usually have a relative lactase insufficiency. Ingested lactose may be fermented into short-chain fatty acids and absorbed. Overproduction or accumulation of short-chain fatty acids in the proximal colon and/or distal ileum may play a role in the pathogenesis of NEC (14). Thus, impaired nutrient digestion, especially lactose, maltodextrin, and cow's-milk protein, coupled with delayed transit time and enteral formula feeding, can expose an immature gut to NEC (15).
DYSBIOSIS IN IMMATURE INTESTINE
An inappropriate composition of gut microflora or an unfavorable balance between commensal and pathogenic bacteria (dysbiosis) is perhaps the most important trigger in the pathogenesis of NEC. PVLBW neonates may be particularly susceptible to dysbiosis. Studies have shown that preterm neonates are at greater risk for developing abnormal intestinal colonization, secondary to contact with nosocomial flora and frequent exposure to antibiotics in the neonatal intensive care unit. Early abnormal stool colonization with Clostridium perfringes is associated with later development of NEC.
Research further demonstrates that some specific events are responsible for the induction of apoptosis in NEC (16). Bacterial invasion and/or local inflammatory cytokines may cause progression of gut injury to necrosis following loss of intestinal barrier integrity with intestinal bacteria translocation (17). Dysbiotic flora with predominant pathogens plays an important role in the pathogenesis of NEC. Researchers have reported an inability to induce severe NEC in animal models without the pathologic flora.
Commensal bacteria can regulate the expression of genes important for barrier function, digestion, and angiogenesis. In vitro studies have demonstrated that many species of commensal bacteria have the ability to dampen the inflammatory response via inhibition of the nuclear factor κ light-chain enhancer of activated B cells (NF-κB) (18,19). It is likely that the balance of pro- and anti-inflammatory signaling is critical in maintaining normal intestinal functions.
IMMATURE INTESTINAL IMMUNITY WITH UNCONTROLLED INFLAMMATION
The immature intestine is thought to respond to injury with excessive inflammation, which is likely to be the final common pathway in the pathogenesis of NEC (20). Inflammation can be initiated by the bacterial cell wall product endotoxin and by ischemia reperfusion.
Many pro- and anti-inflammatory mediators are involved in the pathogenesis of NEC. Proinflammatory mediators and cytokines that are upregulated in infants with NEC include platelet-activating factor (PAF), tumor necrosis factor-α, NO, interleukin-1β (IL-1β), IL-6, IL-8, IL-12, and IL-18, thromboxanes, endothelin-1, and free oxygen radicals. Several anti-inflammatory compounds such as prostacyclin, growth factors (eg, EGF, heparin-binding epidermal growth factor, insulin-like growth factor), erythropoietin (EPO), IL-4, IL-10, glutamine, and arginine can downregulate intestinal inflammation (21). The releases of potent biologically active mediators from immature and damaged GI cells, together with inflammatory cells, amplify the inflammatory response, leading to tissue damage and NEC.
It is difficult to delineate the specific role of each of these mediators, but evidence indicates that the loss of the balance between pro- and anti-inflammatory regulators in favor of proinflammatory response in preterm infants is a critical factor contributing to the final common pathway of NEC (22).
The most important proinflammatory mediator that induces intestinal injury, PAF, is present in the intestinal mucosal environment following synthesis and secretion from epithelial cells, inflammatory cells, and bacteria. Studies in animal models have shown that newborn animals stressed with formula feeding and asphyxia express higher concentrations of PAF receptor than mother-fed controls. Following PAF receptor activation, epithelial cells have accelerated apoptosis, increased mucosal permeability and reduced the integrity of tight junctions, and increased signal transduction that may facilitate the entry of bacterial products such as lipopolysacharide (LPS) from the gut lumen into the tissues, triggering the inflammation cascade (23).
Both PAF and LPS can activate the NF-κB transcription factor that triggers the gene expression of many proinflammatory cytokines (24). Proinflammatory cytokines cause polymorphonuclear activation and tissue inflammation. Neutrophils release inflammatory mediators (25) and cause further intestinal inflammation and necrosis, leading to a vicious cycle.
The exaggerated inflammatory response of the immature intestinal cells to pathogenic stimuli is likely the result of abnormalities and/or immaturity of pattern recognition receptor signaling (26). Toll-like receptors (TLRs) are receptors for LPS on the cell surface that play an important role in initiating the inflammatory and immune defense response. Significantly increased TLR4 mRNA expression has been documented in fetal human intestine (27). Increased TLR4 expression has also been noted in formula-fed and hypoxia-stressed rats (28). When LPS binds to TLR4, abnormal TLR activation (perhaps via the influence of PAF) occurs (29) and a series of chaperone and signal transduction molecules are activated, resulting in NF-κB translocation from the cytoplasm to the nucleus, where this important transcription factor activates the gene expression of multiple proinflammatory cytokines (30).
Thus, the host recognizes the pathogen and initiates steps to eradicate its presence. TLR4 is normally minimally expressed on the intestinal epithelium in adults; however, in the stressed neonatal animal (and probably in humans), epithelial TLR4 expression on the intestines is increased, together with the activation of downstream proinflammatory cytokines (28). Activation of enterocyte TLR4 leads to the development of NEC through profound and deleterious effects on promoting intestinal injury and reducing the capacity for mucosal repair (31,32). Recent experiments have shown that mice with a mutation in their TLR4 gene are resistant to NEC (33).
Gribar et al (31) showed reciprocal patterns of expression of TLR4 and TLR9. TLR9 activation with CpG-DNA, the receptor for bacterial DNA, leads to an inhibition of TLR4-mediated signaling in enterocytes, resulting in attenuation in the extent of LPS-mediated enterocyte apoptosis and a reversal in the inhibition of enterocyte proliferation and migration, thus reducing NEC severity significantly, whereas TLR9-deficient mice exhibited increased NEC severity. Thus, TLR9 can serve as a novel therapeutic approach for infants with this devastating disorder (31,34).
APOPTOSIS INITIATES A VICIOUS CYCLE
Developmental immaturity that leads to inadequate anti-inflammatory response can result in intestinal damage and intestinal epithelial cell death (35). Studies have shown that mice that are null for NF-κB activation in intestinal cells manifest inadequate inflammation response and marked epithelial apoptosis in response to transient hypoxia (36). Intestinal epithelial apoptosis has been increasingly recognized as either an initiating or a critical step in the pathogenesis of NEC (23). The possibility that apoptosis is the earliest stage in the initiation of NEC is supported by experimental and animal studies that demonstrate how agents such as carbon monoxide (antiapoptosis agent) (37), insulin-like growth factor (38), and EGF (12) reduce intestinal apoptosis and attenuate NEC.
Given these data, it can be posited that formula feeding and bacterial invasion (39) induce epithelial apoptosis and impair gut barrier integrity, thereby allowing bacterial translocation and the entry of products such as LPS and PAF into intestinal submucosa. Upregulating intestinal epithelial TLR4 with exaggerated inflammatory cytokines induces further apoptosis (40). In the presence of bacterial proliferation, LPS continuously activates NF-κB and accentuates the inflammatory cascade (41), provoking progressive apoptosis and epithelial barrier damage, permitting further entry of large amounts of bacterial toxins and aggravating bacterial translocation. This results in intestinal ischemia, focal necrosis, sepsis, intestinal perforation, and death.
Some investigators hypothesize that genetic susceptibility as IL-4 receptor α-chain mutant allele and AA genotype in the IL-18 gene promoter at position 607 correlate with NEC. A study on this area may eventually aid in the development of specific preventive strategies (42). Given the complex pathophysiology of NEC, any single gene cannot paint the entire picture.
Transfusion-associated NEC, a new disease entity, refers to the preterm infants who develop NEC within 48 hours after receiving blood transfusion. Anemia potentially compromises mucosal integrity with subsequent poor healing, and this injury may be augmented by unknown factors associated with red blood cells transfusions (43); however, the direction of effect of blood transfusions on the incidence of NEC (more transfusions show lower NEC) demonstrated in randomized controlled trials is the opposite of that seen in observational studies (transfusions are associated with NEC) (44). More investigations are needed to clarify the pathophysiology of this new entity.
PATHOGENESIS IMPLICATIONS FOR PREVENTING
Knowing that formula feeding and bacterial invasion induce intestinal epithelial cell apoptosis that initiates the vicious cycle of inflammatory cascade for NEC in preterm infants, strategies of prevention should focus on preventing preterm birth and bacterial invasion and promoting breast milk feeding. Preventing preterm birth is a difficult task at present. There are some strategies presently available for the primary prevention of NEC, including antenatal glucocorticosteroids, to promote the acceleration of intestinal maturity and cautious feeding strategy when feeding with human milk; however, their efficacy remains debatable. Other interventions such as EGF, EPO, and antitumor necrosis factor-α are promising but lack supporting clinical data.
Probiotics can promote maturation of intestinal function, regulate apoptosis, reduce growth and adherence of potentially pathogenic organisms, enhance the production of anti-inflammatory cytokines and secretory IgA, attenuate the production of nitric oxide, modify enteric carbohydrate fermentation, and increase antioxidant activities. Given their broad range of beneficial effects at various levels of gut function and defense, probiotics may just be the “silver bullet” that prevents NEC.
Significant benefits of probiotics have been confirmed in an updated systematic review and meta-analysis (including trial sequential analysis) reported by Deshpande et al (45). Meta-analysis using a fixed-effect model (11 trials, n = 2176) estimated a lower risk of stage II NEC or greater in the probiotic group (relative risk [RR] 0.35, 95% confidence interval [CI] 0.23–0.55; P < 0.00001). The risk of death (9 trials, n = 2051) was reduced significantly in the probiotic group compared with the control group (RR 0.42, 95% CI 0.29–0.62; P < 0.00001).
Trial sequential analysis has indicated that probiotics reduce the incidence of NEC by at least 30%. Another meta-analysis reported by Wang et al suggest that probiotic supplement was associated with a significantly decreased risk of NEC in PVLBW infants (RR 0.33; 95% CI 0.24–0.46; P < 0 .00001). Risk of death was also significantly reduced in the probiotic group (RR 0.56; 95% CI 0.43–0.73; P < 0.0001). The included trials report no systemic infection with the probiotic supplemental organism (46). Two follow-up studies in up to 3-year-olds did not show any adverse outcomes (47,48).
Despite all of these extremely encouraging results, the presence of clinical heterogeneity, heterogeneity of probiotic strains, reproducibility of results in different setups, and cross-contamination have been cited as reasons for not introducing the routine use of probiotics at this stage in preterm neonates (49). A few case reports of Lactobacillus (50) and 1 report of Bifidobacterium breve sepsis (51; references 51 and 52 can be viewed online at http://links.lww.com/MPG/A251) have raised concerns regarding probiotic sepsis in immunocompromised patients or in those who have underlying medical conditions. Nonetheless, given the health burden of NEC and the fact that many level III neonatal nurseries in Finland, Italy, and Japan have been using probiotics routinely for >1 decade without any reported adverse effects, early oral probiotics with mixed flora is appropriate to prevent NEC.
Amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC (52). Amniotic fluid is abundant in EGF, which is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor. The inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocks the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid does not prevent TLR4 signaling in EGFR- or peroxisome proliferator–activated receptor γ-deficient enterocytes, or in mice deficient in intestinal epithelial EGFR. Purified EGF also attenuates the exaggerated intestinal mucosal TLR4 signaling in wild-type mice.
Moreover, amniotic fluid–mediated TLR4 inhibition reduces the severity of NEC in mice through EGFR activation. Interestingly, NEC development in both mice and humans is associated with reduced EGFR expression that is restored by the administration of amniotic fluid in mice. This also enhances recovery from NEC in humans, suggesting that a lack of amniotic fluid–mediated EGFR signaling may predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this disease.
Considering the difficulties in preventing preterm birth, the single-most important risk factor for NEC, it is expected that the burden of NEC will continue. Strategies such as antenatal glucocorticoids, postnatal preferential use of human milk, and cautious approach to enteral feeding indicate the need for continuous research in these areas. Interventions such as arginine, EGF, and EPO may be promising but warrant further evaluations, including definitive clinical trials. Probiotics seem to be the most significant advance in NEC prevention at present, although there are still important issues (best strains, product availability, doses, duration of prophylaxis, and adverse outcomes) that need to be addressed by future research. Future directions in the field to understand the roles of vascular networks, intestinal stem cells, genetic factors, and the intestinal microbiome are likely to provide tremendous insights into the development of this disease.
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