The role of ischemia and reperfusion injury in the pathogenesis of NEC has largely been characterized through neonatal animal models. The molecular events leading to cellular damage have been well characterized, although the factors initiating this process in the context of clinical NEC are still unknown. Hypoxia at the cellular level leads to a marked increase in the production of xanthine oxidase (XO). Upon reperfusion, XO converts hypoxanthine to xanthine and uric acid, releasing a host of reactive oxygen species in the process (hydroxyl, superoxide, and hydrogen peroxide radicals). These species can induce substantial damage to lipoprotein components of the cell membrane, increasing permeability and facilitating bacteria translocation. Even relatively brief periods of hypoxia can lead to prolonged and substantial changes in mucosal permeability through this mechanism (16).

The precise mechanism and temporal relationship for ischemic injury in the context of NEC remains unclear. Gross and histopathologic findings do not support a thromboembolic process or a generalized low-flow state as a primary mechanism of injury. In this regard, mesenteric infarction and low-output states (previously believed to be associated with NEC through umbilical artery catheter use and cardiogenic shock, respectively) appear to be distinct clinicopathologic entities. Increasing evidence suggests that ischemic injury associated with NEC may result from a maladaptive vascular response to early pathogenic events in the disease. As such, ischemia may play a role in the progression of NEC rather than serving as a critical precipitating factor.

Several theories have been proposed to explain the etiology of ischemia associated with NEC and prematurity. Hyperactive extrinsic vascular regulation (the so-called “diving” or “Herring-Breur” reflex) has been implicated because of its potential relevance to the neonate (17,18). Initially described in diving mammals, the reflex involves a preferential diversion of blood flow to the heart and brain during periods of severe hypoxia. This presumably occurs during the period of parturition. As such, this theory cannot adequately explain the clinical observation that NEC commonly occurs during the second week of life. Furthermore, significant episodes of hypoxia prior to bowel injury cannot be identified in most cases of NEC.

Other studies have attempted to characterize the vasoregulatory responses of the premature gut to hypoxia and prolonged sympathoadrenergic stimulation. Studies using neonatal pigs have identified a maladaptive response to these insults in the way of prolonged and exaggerated vasoconstriction (19). Such responses may exacerbate transient (and likely physiologic) episodes of hypoxia. Potential mechanisms include the presence of labile myogenic vascular reflexes, which may be particularly sensitive to changes in venous pressure (20). Such maladaptive vasoregulatory reflexes are not observed in the intestine of mature swine. Protection appears to be conferred through autoregulatory “escape” mechanisms, which are hardwired into the mature intestine (19). Future studies will need to further characterize how complex vasoregulatory mechanisms regulate the intestinal microcirculation, and how these may become dysfunctional in the premature infant.

Several inflammatory mediators are elevated in the serum of premature neonates with NEC. These include tumor necrosis factor-alpha (TNF-α), platelet-activating factor (PAF), and the interleukins 6 and 8 (IL-6 and IL-8), among others (21,22,23). Intestinal cells of premature infants appear to elaborate higher concentrations of proinflammatory cytokines (particularly IL-8) in response to endotoxin and IL-1 compared with mature cells (21). The primary challenge has been to distinguish between those that play a pivotal role in the development and progression of NEC, and those that are nonspecific markers of inflammation. This is particularly challenging given the complexity and
redundancy of the inflammatory cascade. Furthermore, many conditions associated with NEC can increase serum levels of inflammatory mediators through independent mechanisms (e.g., sepsis).

Data from animal models have identified PAF as a leading candidate for initiating the early pathogenic events in NEC. PAF is a potent vasoconstrictor of the mesenteric circulation and increases mucosal permeability (24). Infusion of PAF into the intestines of neonatal pigs leads to injury resembling NEC, and will also exacerbate the severity of injury in response to ischemia-reperfusion challenge (25,26). Pretreatment with PAF antagonists (WEB-2086) significantly attenuates injury in both models (27,28). These observations suggest that PAF may be integral to the pathogenic changes seen with ischemia-reperfusion injury. PAF may also influence the progression of NEC through secondary inflammatory pathways (particularly TNF-α) and as a potent chemokine for neutrophil activation (25,26). The propensity of NEC to involve the distal small bowel may reflect the relatively high concentration of PAF receptors in this area (29).

PAF-acetylhydrolase is the enzyme responsible for the metabolism of PAF, and serum levels of this enzyme are markedly attenuated in prematurity (30). Levels are even lower in premature neonates diagnosed with NEC. PAF-induced injury may therefore be due to an increase in functional activity rather than overproduction. The deleterious effects of PAF may be dependent on the presence of other factors, including bacterial endotoxin (31,32). Intestinal injury is not observed when PAF is given to germ-free rats (32). Furthermore, injury to mucosal cells in the presence of bacterial endotoxin may likewise be dependent on the presence of PAF. Pretreatment with PAF antibodies significantly reduces injury following the infusion of endotoxin (lipopolysaccharide) into the guts of neonatal pigs (33).

Nitric oxide (NO) has received much attention for its apparent protective function during ischemic stress. Endogenous NO is produced by three different isoforms of the enzyme nitric oxide synthase (NOS). Sources include endothelial cells, phagocytic cells in the circulation and tissues, and others. The protective effects of NO are mediated through vascular smooth muscle relaxation. This property may be important for counteracting the influence of vasoconstricting cytokines during periods of inflammation and ischemia. NO may also protect mucosal cells by directly modulating PAF activity and limiting neutrophil adhesion to the vascular endothelium (34).

Evidence for the protective role of NO is found in several observations from animal models: (1) The severity of mucosal injury is inversely related to the activity of NOS in tissue preparations; (2) inhibitors of NOS such as L-arginine significantly worsen PAF-induced intestinal necrosis; and (3) NO donors significantly reduce PAF-induced intestinal necrosis (35,36,37). These observations led to the postulate that the NOS regulatory pathway may be dysfunctional in premature neonates. However, the activity of NOS was found to be increased in premature neonates with documented NEC (38,39). It is possible that this observed increase could be inappropriately low when compared with a similar response in mature animals. However, more recent laboratory studies have suggested that the role of NO in the context of NEC may not always be protective. Higher activity levels of the inducible form of NOS have been correlated with increased severity of injury in the neonatal rat model (39). The reaction between NO and reactive oxygen species to generate even more reactive peroxynitrate compounds has been postulated as a potential mechanism for injury. These observations suggest that, much like molecular oxygen, the physiologic effects of NO may depend on the local cellular milieu. Further studies need to clarify the precise role of NO in the context of NEC, and how this protective mechanism may be exploited for potential therapeutic strategies.

Bacteria appear to be critical in the pathogenesis of NEC, given the observation that the disease does not occur before intestinal colonization is established. The degree of bacterial overgrowth in NEC appears to exceed that observed with other diseases associated with intestinal necrosis (14). Furthermore, the pathologic finding of pneumatosis in association with intestinal necrosis is exceedingly rare, except in the setting of NEC. Pneumatosis results from the accumulation of hydrogen gas in the intestinal wall due to fermentation of carbohydrates by intestinal flora. With stasis and significant bacterial loads, intraluminal pressure may increase to the point of impeding venous return, thereby promoting ischemia. However, the ability of colonizing bacteria to ferment lactose has not been correlated with the development of NEC. Furthermore, evidence for bacterial overgrowth is absent in up to one-third of pathology specimens (14).

These observations suggested that other bacterial factors may be important for initiating the pathogenic changes in NEC. Certain strains of Escherichia coli and Clostridia can cause intestinal necrosis directly by the elaboration of potent exotoxins (40). These organisms have been identified in relative “epidemics” of NEC affecting NICUs (41). Such epidemics have been halted with the implementation of organism-specific antibiotics and the appropriate hygiene measures. However, cultures obtained from blood, stool, and peritoneal aspirates in most cases of spontaneous NEC do not reveal pathogenic organisms. The most commonly isolated species include E. coli, Klebsiella, Enterobacter, Pseudomonas, Clostridium, and coagulase-negative Staphylococci, among others (42,43). This would suggest two different mechanisms of injury with a shared final common pathway. It is likely that, in true cases of NEC, normally nonpathogenic bacteria become
functionally pathogenic due to changes in the mucosal barrier that occur early in the course of disease.

Several physiologic deficiencies have been characterized in the gut of premature neonates (Fig. 79-1). Peristalsis and other aspects of intestinal motility can be substantially compromised, particularly when neonates are born prior to their eighth month of gestation (44). Clearance of bacteria may be impeded, potentially leading to stasis and bacterial overgrowth. Gut-associated immunity may also be affected. Relatively fewer functional B lymphocytes are present in the immature gut, and the ability to produce sufficient amounts of secretory IgA is reduced (45). This antibody is believed to protect mucosal cells by preventing the binding of pathogenic organisms. The production of pepsin, gastric acid, and mucus are also decreased in prematurity (46,47). These factors may have important secondary roles in limiting the proliferation of intestinal flora and their ability to bind to mucosal cells. Intestinal trefoil factor is a peptide that has recently been characterized in the mature rat intestine (48,49). The peptide appears to have several adaptive functions, including protecting the mucosal barrier from microbial invasion and limiting the production of reactive oxygen species during ischemic stress. Significantly lower levels of mRNA for this peptide have been found in the prenatal rat. In concert with potentially dysfunctional vasoregulatory mechanisms, these characteristics may collectively act to lower the threshold for NEC in premature infants.

Epidemiologic studies have identified feeding as an important factor in the development of NEC. The majority of neonates who develop this disease have been previously fed, and the relative incidence of NEC is as much as 50% higher in this cohort (9). The relationship between feeding and NEC is complex, and appears to be dependent on the both the composition and quantity of substrate. Infusion of highly concentrated synthetic formulas leads to spontaneous endotoxemia and increased levels of PAF in healthy preterm infants (50,51). Other studies have demonstrated that high-osmolarity formulas can directly injure the villus border of mucosal cells (46). Malabsorption is common in premature neonates, and high concentrations of undigested fatty acids may also contribute to mucosal injury. Increased production of hydrogen gas from the fermentation of large carbohydrate loads may occur in the presence of stasis and bacterial overgrowth. Multiple clinical series have sought to determine whether the rate with which feedings are advanced in premature infants relates to the occurrence of NEC. Results have been inconclusive. Refer to the “Prevention” section later in this chapter.

Clinical and laboratory studies have identified a multitude of pharmacologic agents as possible risk factors for NEC. Case-control studies have shown an increased risk for NEC in neonates exposed to indomethacin both postnatally and prenatally (maternal tocolysis) (46,52,53). Indomethacin blocks prostaglandin synthetase and has been shown to impair mesenteric blood flow by increasing mesenteric vascular resistance (54). However, an association suggesting causality in the development of NEC has not held up in more rigorous clinical studies (55).

Other pharmacologic agents have been proposed to cause NEC through a variety of mechanisms. Methylxanthine compounds increase bacterial loads by altering gut motility and can damage enterocytes directly by their intraluminal metabolites (56). Vitamin E has been shown to interfere with intracellular bacterial killing in phagocytic cells (57). The hyperosmolarity of some drug preparations has been postulated to injure the villus border of mucosal cells (58). This potential mechanism is less plausible, however, given the relatively small volumes associated with clinical doses for most agents. Vasopressors and other agents used for hemodynamic support may exacerbate intestinal injury through their effects on the mesenteric circulation. Despite these potential mechanisms, no good clinical evidence exists to suggest that any of these agents play an important role in the initiation of NEC.

Case-control studies have identified relatively weak and inconsistent associations for other potential risk factors. These include hypoglycemia, premature rupture of membranes, chorioamnitis, hypercoagulable conditions, low-output states, prenatal cocaine exposure, and the use of umbilical catheters, among others (59,60,61,62,63,64). The intestinal injury associated with many of these “risk factors” is likely mediated through thromboembolic or vasoconstrictive mechanisms. Although clinically apparent injury may occur through the same final common pathway (ischemia and reperfusion), the initial pathogenic events for what we define as NEC may be quite different. This may suggest the misclassification of disease (and associated risk factors) in many earlier observational studies. Furthermore, the interpretation of existing clinical studies can be challenging for a number of other reasons. These include the frequent use of retrospective study designs, the frequency and severity of comorbid conditions, and difficulty with modeling the ever-increasing complexity of the neonate–NICU interaction, among others.

Our understanding of the pathophysiology of NEC is improving at a rapid pace. The experimental and clinical evidence outlined previously has led to a better understanding of the events that may be important for the initiation and progression of disease. Although the precise nature and temporal relationship of these events are yet to be determined, a unifying concept of pathogenesis is emerging from currently available data.

The initial insult leading to NEC may be a subclinical event such as a brief episode of perinatal hypoxia or postnatal infection. The injury from this event may not be clinically relevant in neonates with normally developed intestines. Upon colonization of the gut, bacteria may bind to injured mucosal cells and elicit a localized inflammatory response in reaction to endotoxin and other bacterial products. This may lead to further inflammation as the affected endothelial cells release proinflammatory cytokines, including TNF-α and PAF. In the presence of abnormal counterregulatory mechanisms (e.g., reduced PAF-acetylhydrolase), these factors may increase the permeability of mucosal cells leading to the translocation of bacteria and bacterial products. The inflammatory response is then further amplified, leading to the recruitment and activation of circulating neutrophils. Activation of neutrophils may further injure damaged mucosa through the release of secondary mediators and reactive oxygen species. A maladaptive vasoconstrictive response may then follow, further exacerbating injury through ischemia and reperfusion. The end result is a vicious cycle of positive feedback mechanisms that can ultimately lead to frank necrosis and perforation. This process may evolve over days, with injury and repair maintained in a fine balance. Other factors that may compromise mesenteric perfusion (sepsis) or increase oxygen demand (feeding) may tip the balance toward progressive and irreversible injury.

NEC must be differentiated from other conditions associated with abdominal distention and sepsis. The most common challenge faced by the clinician is differentiating NEC from sepsis of a different etiology associated with ileus. Early NEC and sepsis with ileus can appear clinically indistinguishable. In many cases, differentiation between the two is only possible after observing the course of the disease in each patient.

The term focal intestinal perforation (FIP) has been applied to the surgical finding of a single isolated perforation occurring in a premature infant that is not associated with the radiographic finding of pneumatosis. Much debate has ensued as to whether FIP is simply a “mild” version of NEC or whether it is a distinct disease entity. Clinical studies have observed that perforations associated with FIP occur relatively earlier than NEC, and the lesions occur almost
invariably on the antimesenteric side of the distal ileum (68,69). Some reports have also described a different histologic profile associated with FIP, with coagulation necrosis and pneumatosis being notably absent in most specimens (68,69). The histology of NEC is very nonspecific, however, and early lesions may not exhibit the classic findings of well-established NEC (following secondary ischemic injury). Patients with FIP clearly have a better prognosis than those with NEC, although this may simply reflect the difference in the severity of bowel necrosis in the two cohorts. Ultimately, there may be little clinical relevance in distinguishing these processes, as the principles of management are the same for both.

Laboratory parameters in patients with NEC are often nonspecific and generally indicate the presence of an inflammatory condition. Leukocyte and platelet counts may be elevated, normal, or low, depending on the severity of NEC and associated sepsis. Leukocytosis is the most common abnormality in NEC and is frequently accompanied by a refractory metabolic acidosis (65). Anemia may also be present if there is significant hemorrhage from the bowel wall. Severely depressed leukocyte and platelet counts have been associated with advanced disease and a worse overall prognosis (70,71). However, none of these individual parameters have sufficient sensitivity, specificity, or predictive accuracy to be of useful diagnostic value.

More recent efforts have attempted to identify novel serum markers for the earlier diagnosis and treatment of NEC. Neonates with confirmed stage II and III NEC were found to have significantly elevated serum levels of PAF compared with age-matched controls without the disease (72). However, only 3 of 11 (27%) patients in this case-control study had elevated levels prior to the onset of clinical signs. Furthermore, the lack of data regarding PAF levels in other causes of abdominal sepsis brings into question the specificity of this assay for NEC. Serum fatty acid-binding proteins have also been examined as a potential diagnostic marker for NEC. Similar to the PAF assay, levels were elevated in only a small proportion (12.5%) of patients with stage I disease (73). Further study is required to better define the diagnostic utility of these assays.