Ischemic Injury

Ischemic injury is thought to play a central role in the cascade of events leading to the development of NEC. Several investigators have used animal models of NEC that rely on the induction of intermittent ischemia and reperfusion of the intestine, typically through temporary occlusion of the blood supply to the small intestine. Although such models may provide valuable information regarding the biology of intestinal ischemia reperfusion injury, it is noteworthy that a clear ischemic–reperfusion injury is often absent or very remote in patients in whom NEC develops during the convalescence phase of their hospitalization.

Neonatal asphyxia, recurrent episodes of hypoxia–bradycardia, systemic arterial hypotension, patent ductus arteriosus, congenital heart disease, and polycythemia are some of the mechanisms that may lead to intestinal ischemia. In states of hypoperfusion, the “diving reflex” is activated as a protective mechanism to ensure adequate blood supply to vital organs, including the brain and the heart, while shunting blood from the splanchnic circulation, thus worsening gut ischemia. Ischemic injury of the intestinal mucosa ensues, followed by activation of the inflammatory cascade, reperfusion injury, and gut barrier dysfunction with bacterial translocation and the systemic effects associated with it. Basal intestinal vascular resistance is maintained by a dynamic balance between constrictor (endothelin-1 [ET-1]) and dilator (NO) stimuli, and in newborns, this balance favors NO-dependent vasodilation, leading to a low vascular resistance ensuring adequate blood flow to the rapidly growing intestine. Disruption of intestinal endothelial cell function could alter this balance favoring ET-1-dependent vasoconstriction and thus lead to significant intestinal ischemia. Doppler studies of the superior mesenteric artery in premature infants who later developed NEC demonstrated a high resistance pattern suggestive of abnormal intestinal blood flow. Decreased resistance to blood flow was found in patients with established NEC. It has been postulated that this apparent discrepancy may actually represent a biphasic response of the mesenteric vessels, with an initial phase (high resistance to flow/ischemic injury), followed by rebound hyperemia (low resistance to flow/reperfusion injury), due to the release of inflammatory mediators. It is possible that alterations in the autoregulation of intestinal blood flow, mediated by a balance between vasoconstrictor and vasodilator molecules, play a role in the development and progression of NEC. In addition, intestinal tissue removed from infants with NEC demonstrates abnormalities in expression and regulation of these important vasoactive mediators.

Gut Barrier

In mature animals, the intestine has many physical barriers to bacteria, including peristalsis, gastric acidity, proteolytic enzymes, intestinal mucus, cell-surface gly­coconjugates, and tight junctions between intestinal epithelial cells. These protective mechanisms limit the bacterial microenvironment to the gut lumen and prevent attachment and translocation across the intestinal epithelium. However, experimental data demonstrate that pathogenic organisms adhere to and translocate across the intestine to a greater extent in immature compared to mature animals. Abnormal peristaltic activity and hypomotility in immature infants may increase bacterial adherence, allowing for bacterial overgrowth. In the NICU, the use of narcotics for sedation and pain management may accentuate this phenomenon. In preterm infants, the repertoire of cell-surface glycoconjugates, which serve as adhesion sites for a variety of microbes, have a different pattern of carbohydrate residues. This functional difference may in part account for differences in bacterial colonization patterns in these infants. Colonization of the intestine by commensal microorganisms is a key step in intestinal maturation and maintenance of the gut barrier. Alternatively, colonization with pathogenic organisms may trigger an inappropriate reaction by the immature intestine with activation of the inflammatory cascade, which promotes disruption of the gut barrier resulting in NEC. Human defensins (or cryptidins) produced and secreted from Paneth cells protect against bacterial translocation; however, this mechanism is impaired in premature infants, particularly those with NEC. Trefoil factor peptides (TFFs, 1 to 3) are part of the protective mechanism operating in the intestinal mucosa and play a fundamental role in epithelial protection, repair, and restitution. These secreted peptides have been identified in a site-specific pattern in the gastrointestinal mucosa, and their expression has been shown to be upregulated in early stages of mucosal repair. A study by Lin et al. demonstrated that TFF3 mRNA and protein expression are deficient in immature rats. Impaired mucosal regeneration in part due to failure of upregulation of TFF peptide expression may contribute to the pathogenesis of NEC. Tight junctions between epithelial cells maintain the semipermeable properties of the intestinal epithelium, limiting the passage of bacteria and other macromolecules. Immaturity in the composition and function of the tight junctions through the interactions of structural proteins (claudins and occludins) may explain the increased permeability of the immature intestine. Furthermore, changes in expression of genes coding for these structural proteins have been described during the lesional and reparative phases of NEC.

In premature infants, there is immaturity of the functional barrier that limits growth of bacteria, the immunologic host–defense mechanism, and various biochemical factors involved in gut barrier protection. It is known that the numbers of intestinal B and T lymphocytes are decreased in neonates and do not approach adult levels until 3 to 4 weeks of life. Newborns also have reduced levels of secretory immunoglobulin A (IgA) in salivary samples, presumably reflecting decreased activity in the intestine. Furthermore, the intestinal lamina propria is devoid of IgA-secreting cells, rendering the newborn more susceptible to infection on the mucosal surface.

Bacterial Colonization

The role of bacterial colonization in the pathogenesis of NEC has long been recognized. In utero, the intestinal tract is free of bacteria, but it becomes rapidly colonized during the first days of life. Mode of delivery, treatment with antibiotics, and type of enteral feedings affect the timing and patterns of colonization. Infants delivered vaginally have earlier colonization with both Bifidobacterium and Lactobacillus , whereas infants delivered by cesarean section can have colonization with these beneficial organisms delayed by up to 30 days. After birth and during the first week of life, the normal breast-fed newborn gut is colonized by predominantly protective anaerobe bacteria including lactobacilli and bifidobacteria. In formula-fed infants, similar numbers of Bacteroides and Bifidobacterium are found with minor components of the more pathogenic Staphylococcus , Escherichia coli , and Clostridium . In contrast, the preterm infant’s gut is exposed to a flora with a predominance of potentially pathogenic gram-negative species, with a lesser degree of bacterial diversity and fewer protective anaerobe species. The use of broad-spectrum antibiotics during the immediate neonatal period may select a population of resistant organisms, thereby aggravating this situation. In the presence of other risk factors, alteration of the intestinal microbiome may set the stage for the later development of NEC. Despite the description of outbreaks of NEC in some nurseries, no single specific organism can be identified as the etiologic agent. Klebsiella , E. coli, Clostridium species, and Staphylococcus epidermidis, as well as a variety of viruses and fungal species, have all been isolated from patients with NEC. Particular interest has been placed on Enterobacter sakazakii (ES), which can cause sepsis and meningitis in low-birth-weight infants and has been identified in the hospital environment in association with infant bottle brushes and food preparation equipment. Ingestion of ES-contaminated infant formula has been implicated in cases of necrotizing enterocolitis. In vitro and in vivo studies suggest that ES adheres to the enterocyte and promotes cell necrosis and apoptosis in a dose-dependent manner. In 2002, the U.S. Food and Drug Administration (FDA) published a warning regarding the presence of ES in baby formula.

Feedings and NEC

The contribution of different feeding practices to the pathogenesis of NEC has been extensively studied. Timing of introduction of feeds, type, volume, and rate of advance have all been the subject of clinical studies. Experimental data suggest that introduction of feeds is important to establish intestinal barrier function. In a rat model, starvation reduces gut mucosal barrier function; conversely, early feeds improved intestinal mucosal growth in piglets. It has been shown that infants who were never fed and developed NEC were more likely to rapidly progress to severe disease. It is possible that the early introduction of breast-milk feedings prepares the intestinal barrier to better respond to noxious stress later on.

In several randomized controlled trials, rapid advancement of enteral feeds was associated with a shorter time to full feeds and more rapid attainment of birth weight without an increased incidence of NEC. These results are tempered by another trial where rapid progression of enteral feeds was associated with NEC. There have been several Cochrane reviews addressing timing of feeding introduction, rate of feeding advancements, and enteral fasting versus introduction of early trophic feeds. None of those interventions seems to decrease the incidence of NEC, whereas more conservative feeding schedules result in delays to achieve full feeds and to regain birth weight. Breast milk or donor breast milk is preferred for initiation of enteral alimentation for preterm infants when available. A protocol of enteral feedings containing at least 50% of human milk in the first 14 days of life was associated with a sixfold decrease in the incidence of NEC. Of interest, the benefits of breast-milk feedings may follow a dose-related beneficial effect on the risk of NEC and mortality. In addition to the well-known immunologic properties in breast milk, the presence of other factors such as epithelial growth factor (EGF), erythropoietin, insulin-like growth factor (IGF), and the anti-inflammatory cytokine IL-10 may account for its protective effects. The currently available data support the promotion of breast milk as the initial nutrient of choice for the premature infant. It is well known that feeding practices vary among centers, but in general, judicious introduction of gut-priming feeds followed by a steady rate of advancement with close attention paid to feeding intolerance seems to be the preponderant approach. The use of standardized feeding protocols has been shown to be protective. The effects of other enterally administered supplements (e.g., vitamins, iron, fortifiers) on the incidence of NEC are unclear.

Abnormal Inflammatory Response

The modulation of the inflammatory response seems to be developmentally regulated, and it requires an appropriate balance between proinflammatory and anti-inflammatory signaling in order to maintain a healthy intestinal homeostatic environment.

The inability of the premature newborn to distinguish between and respond appropriately to commensal and pathogenic microorganisms and the aberrant regulation of other mediators such as platelet-activating factor (PAF) lead to a proinflammatory environment that could contribute to the pathology seen in NEC. Human fetal intestinal epithelial cells demonstrate an exaggerated production of inflammatory cytokines in response to pathogenic and commensal bacteria, as well as to endogenous inflammatory mediators such as TNFα and IL. Several proinflammatory cytokines that mediate inflammatory cell recruitment through activation and amplification of the immune response in local host defense have been implicated in NEC, including TNFα, IL-1β, IL-6, IL-8, IL-12, and IL-18. Anti-inflammatory cytokines modulate the host’s inflammatory response; a lack of upregulation of these molecules to counteract the effects of proinflammatory mediators results in more severe tissue injury. Both anti-inflammatory cytokines, IL-4 and IL-10, have been implicated in NEC.

Lipopolysaccharide (LPS), which is a principal component of the outer cell wall of gram-negative bacteria, recognizes and binds to Toll-like receptor 4 (TLR4). Circulating LPS is increased in patients with NEC, which inhibits epithelial restitution and initiates inflammatory signaling cascades within the enterocyte, including activation of the transcription factor nuclear factor (NF)-κB. NF-κB proteins activate transcription of a wide variety of genes important in inflammatory and immune responses. In the resting state, NF-κB dimers are bound in the cytoplasm to inhibitory κB (IκB) proteins. By activation of an IKK kinase, NF-κB is freed to translocate to the nucleus, where it triggers gene transcription. An autoregulatory feedback loop exists in which NF-κB activation leads to IκBa synthesis, which in turn can terminate the NF-κB response. However, it has been shown that immature enterocytes have increased NF-κB activity associated with decreased baseline expression of IκB isoforms. In addition, there is more rapid IκBa degradation and delayed resynthesis of IκBa in immature enterocytes. These data suggest that the increased inflammatory cytokine production in the immature intestine may result from the combined effect of increased NF-κB activity and translocation to the nucleus because of decreased IκB expression, and decreased inhibition of NF-κB due to an accelerated degradation and delayed resynthesis of IκBa.

The pathogenesis of NEC is complex, and our understanding of it is still incomplete. Multiple pathogenic mechanisms affecting the premature infant, such as perinatal ischemia, feedings and bacterial colonization, and inflammation, converge into a final common pathway leading to intestinal barrier failure, tissue damage, and organ system failure ( Figure 45-4 ). Increasing our understanding of the mechanisms involved in NEC through well-designed clinical and basic research will allow us to develop and implement interventions aimed at early diagnosis and prevention. Currently, introduction of enteral feedings with breast milk is the most readily available way to decrease the incidence of the disease.

The complex interplay of various pathogenic mechanisms affecting the premature infant and converging to a final common pathway leading to necrotizing enterocolitis (NEC).