94 Neonatal Necrotizing Enterocolitis

MICHAEL CAPLAN

Neonatal necrotizing enterocolitis (NEC) is a common

and devastating gastrointestinal emergency that primarily
afflicts premature infants in neonatal intensive care units
(NICUs) worldwide. Despite advances in neonatal care
and significant clinical and basic science investigation,
the etiology remains incompletely understood, specific
treatment strategies are lacking, and morbidity and
mortality from this disease remain high. This chapter
reviews the epidemiology and classic clinical features of
NEC, and describes the current understanding of the
pathophysiology as well as new exciting approaches to
prevention.

Epidemiology
The incidence of NEC varies among US centers and across
continents, but ranges between 3% and 28%, with an
average of approximately 6% to 10% in infants born
weighing less than 1500 grams. There is an inverse correlation between gestational age/birth weight and incidence of NEC; the incidence increases dramatically in the
smallest and most premature infants, and intrauterine
growth restriction confers a higher risk of disease than
that of a normally grown preterm infant. Although there
appears to be a slightly increased prevalence in boys,
some data suggest higher NEC rates in African Americans
compared with whites or Hispanic neonates,79 and this
ethnic difference could be explained by some recently
identified genetic polymorphisms.63,81 Most (90%-95%)
preterm infants who develop NEC are previously fed,
although the onset of disease may be several weeks after
enteral nutrition begins. There is increasing evidence that
human milk feeds may reduce the incidence of NEC. In
the California Perinatal Quality of Care Collaborative,
NEC rates decreased from 7.0% to 2.4% with increasing
rates of human milk feeds.52 Although most neonates
who develop NEC are preterm, 5% to 10% of cases occur
in babies born greater than or equal to 37 weeks gestation. In this population, NEC is almost always associated
with a specific risk factor such as asphyxia, intrauterine
growth restriction (IUGR), polycythemia/hyperviscosity,
exchange transfusion, umbilical catheters, gastroschisis,
congenital heart disease, or myelomeningocele. In these
situations, overt intestinal ischemia is often suspected
and therefore the pathophysiology may differ from that
in the preterm neonate with NEC. Despite significant
advances in neonatal care, the mortality resulting from
NEC has not improved over the last three decades, with
reports of NEC mortality up to 40%.37

Clinical Features
PRESENTATION
Necrotizing enterocolitis can present with a variety of
symptoms and signs; preterm neonates may demonstrate
symptoms of hematochezia, emesis and/or increased
gastric residuals, abdominal distention, lethargy, and
apnea and bradycardia, and signs of neutropenia, thrombocytopenia, metabolic acidosis, tachycardia, abdominal
tenderness, abdominal discoloration, respiratory failure,
and if severe, shock. Guaiac-positive stools are quite
common in nasogastric tubefed preterm neonates (60%75%) and therefore, are not a useful indicator of NEC.
Feeding intolerance occurs frequently in this population
of preterm neonates, but studies indicate that intolerance
is not a reliable marker for the development of intestinal
injury.

DIAGNOSIS
The diagnosis is typically made by the identification of
pneumatosis intestinalis (air in the bowel wall) and/or
portal venous gas on abdominal radiograph, although in
some cases of NEC, commonly in unfed patients, pneumatosis is not appreciated (Figure 94-1). In these situations, NEC may be diagnosed surgically or pathologically,
or in some instances by ultrasound appreciation of portal
venous air. Bell and colleagues suggested a classification
scheme that differentiates suspected NEC (stage I) from
proven NEC (stage II) and advanced NEC (stage III with
peritonitis and/or perforation) (Table 94-1).4 In this
scheme, stage I NEC includes mild systemic signs, abdominal distention with changes in feeding intolerance, but
no confirmatory radiographic evidence. Stage II or proven
NEC has similar symptoms or signs with pneumatosis
and/or portal venous gas, and stage III demonstrates significant systemic signs with radiographic evidence of
intestinal perforation (pneumoperitoneum). This classification scheme is useful in occasional circumstances,
especially when one analyzes studies that evaluate NEC;
readers should be wary of interventions that appear to
influence stage I, suspected disease without altering definitive NEC.

TREATMENT
No specific treatment approaches have influenced the
outcome of NEC, and as such, interventions are supportive and include fluid resuscitation, withholding
feedings with gastric decompression, antibiotics to cover
likely enteric pathogens, correction of acidosis, anemia,
1423

1424 PART 15 THE GASTROINTESTINAL TRACT

A
B
Figure 94-1 Radiologic evaluation of NEC. A, Portal venous gas and pneumatosis intestinalis. B, Perforation with free air on supine film. C, Free
air on cross table lateral.

TABLE 94-1

Modified Bell Staging Criteria for Necrotizing Enterocolitis

Stage

Classification

Signs

Radiologic Signs

I
II

Suspected NEC
Proven NEC

III

Advanced NEC

Abdominal distention, bloody stools, emesis/gastric residuals, apnea/lethargy

Above with: abdominal tenderness metabolic acidosis and
thrombocytopenia
Above with: hypotension, significant acidosis, thrombocytopenia/DIC,
neutropenia

Ileus/dilatation
Pneumatosis intestinalis and/or
portal venous gas
Above with pneumoperitoneum

Modified from Kanto WP, Hunter JE, Stoll BJ. Recognition and medical management of necrotizing enterocolitis. Clin Perinatol. 1994;2:335-346.

and thrombocytopenia as needed, and blood pressure

support. Although blood cultures are positive in approximately 30% of NEC cases and are thought to reflect
a breakdown in the mucosal barrier leading to bacterial translocation, intraluminal enteric bacterial pathogens are thought to contribute to the pathophysiology.48
Antibiotic coverage in this condition typically includes
ampicillin and an aminoglycoside or third-generation
cephalosporin, although occasionally Staphylococcal
species are heavy colonizers of the intestinal tract, and
nafcillin or vancomycin should be considered. In situations with suspected or proved intestinal perforation,
aggressive anaerobic coverage with clindamycin is often
added. Although routine treatment usually proceeds for
7 to 10 days with NPO and antibiotics in uncomplicated,
medical NEC, length of treatment has not been carefully
studied, and one report has suggested earlier refeeding
after ultrasonographic evidence of portal venous air has
resolved.10
Surgical intervention for NEC is required in 30% to
50% of cases reported, although the approach and timing
of these procedures remain controversial. Most physicians agree that intestinal perforation in a NEC patient
requires surgery, but based on three clinical trials, patients

treated with a bedside drain had similar rates of death

and short-term intestinal function as those undergoing
definitive laparotomy.9,64,77 In surgical trials for NEC
treatment, almost 50% of patients treated with a drain
never required a second procedure. In patients without
perforation, but with worsening disease as manifested
by abdominal discoloration and distention, persistent
thrombocytopenia and acidosis, and respiratory failure,
exploratory laparotomy is often undertaken to remove a
discrete segment of necrotic bowel or to confirm the viability of enough remaining intestine to sustain life. Nonetheless, the timing and utility of these procedures in these
complex patients have not been adequately studied.

OUTCOME
Approximately 30% of patients with radiologic evidence
of pneumatosis intestinalis have mild disease and require
a period of bowel rest, but no surgical intervention.
Another 30% of patients eventually succumb to the
disease, with most presenting acutely with rapid deterioration and death. Survivors from NEC have a significant
risk for intestinal stricture; in some reports, as many as
25% develop partial bowel obstruction weeks or months
following the initial presentation. Some patients develop

94 Neonatal Necrotizing Enterocolitis

short bowel syndrome from NEC; postsurgical patients

have an incidence as high as 11% of short bowel syndrome, and these patients are an extremely difficult group
for whom to care. Novel medical and surgical interventions have made only modest improvements on the
morbidity and mortality associated with this dreaded
complication. Of concern, accumulating data suggest that
the neurodevelopmental outcome of NEC patients is significantly worse than their gestational age and birth
weightmatched controls with similar respiratory disease.
The morbidity includes mental retardation as well as an
increased incidence of cerebral palsy, hypothetically
related to white matter injury from cytokine mediators
involved in the systemic inflammatory cascade. Further
studies are needed to confirm this association and clarify
the significance of these important results. Nonetheless,
NEC is a major financial burden nationwide, with
increased initial hospital costs (owing primarily to longer
length of stay) of $60,000 for a case of medical NEC and
up to $200,000/patient for surgical disease.8 Based on the
current epidemiology, this projects an annual cost burden
of $1 billion to the US health care industry without
taking into account the long-term care issues associated
with impaired survivors.

PATHOLOGY
Clues to the etiology are suggested by the pathologic
changes observed in surgical specimens and autopsy
material, including coagulation necrosis (suggesting
some component of ischemic injury), inflammation
(acute and/or chronic), and less commonly, ulceration,
hemorrhage, reparative change, bacterial overgrowth,
edema, and pneumatosis intestinalis.

Pathophysiology of
Necrotizing Enterocolitis
Although the specific etiology of NEC is still controversial, epidemiologic analyses of this disease have identified
strategic risk factors of prematurity, enteral feeding, intestinal ischemia/asphyxia, and bacterial colonization.
Recent studies have begun to delineate the mechanisms
that link these risk factors to the final common pathway
of bowel necrosis.14 It has been suggested that altered
patterns of intestinal colonization initiate an unbalanced
proinflammatory cascade, resulting in intestinal injury
and in many cases, the systemic inflammatory response
syndrome.

PREMATURITY
Greater than 90% of NEC cases occur in premature
infants; there is consistently a higher risk with lower gestational age and birth weight,56 and as such, prematurity
is the most consistent and important risk factor. Although
there are many differences between preterm and full-term
neonates, the specific underlying mechanisms responsible for this predilection of NEC in the premature condition remain incompletely elucidated. Studies in humans
and animals have identified alterations in multiple components of intestinal host defense,92 motility,5 bacterial
colonization,95 blood flow regulation,71 and inflamma-

1425

tory response13,27,67 that may contribute to the development of intestinal injury in this unique population.

ENTERAL FEEDING
Because most cases of NEC occur after feedings have been
introduced (>90%), enteral alimentation is a significant
risk factor for disease in premature infants. Historical
reports identified the onset of NEC several days following
the first feed, but in studies of extremely low birth weight
infants, NEC may be diagnosed several weeks after initiating enteral supplementation.11 This change may reflect
current neonatal practice that typically uses early trophic
or hypocaloric feedings, characterized by small volumes
and slow rates of increase, without a significant impact
on the development of NEC. Although the precise relationship between enteral feedings and NEC remains
poorly understood, studies have identified the importance of breast milk (versus formula), volume and rate of
feeding advancement, osmolality, and substrate fermentation as important factors.
Breast milk feeding appears to reduce the incidence of
NEC in human studies and in carefully controlled animal
models.16,56 Breast milk contains multiple bioactive
factors that influence host immunity, inflammation, and
mucosal protection, including secretory IgA, leukocytes,
lactoferrin, lysozyme, mucin, cytokines, growth factors,
enzymes, oligosaccharides, and polyunsaturated fatty
acids, many of which are absent in neonatal formula
preparations (Table 94-2). Specific intestinal host defense
factors acquired from breast milk such as EGF, PUFA,
PAF-acetylhydrolase, IgA, and macrophages are effective
in reducing the incidence of disease in animals,17,27,55 and
some have been effective in limited human trials.20,29
Nonetheless, breast milk is not completely protective
against NEC in premature infants; the largest prospective
trial identified a reduction by 50% in most birth weight
specific groups, although there was not a statistically significant reduction in disease observed in a randomized
subset from this cohort.56 Because of ethical considerations, it seems unlikely that such an investigation will
be accomplished, although there is renewed interest in

TABLE 94-2

Molecule
IgA, IgG
Leukocytes
Oligosaccharides
PUFA
Lactoferrin
Glutamine
Arginine
PAF-AH
EGF
IL-10
Erythropoietin

Biologic Factors in Breast Milk

That May Influence Necrotizing
Enterocolitis Pathophysiology
Effective in
Animal Model

Effective in
Human Trial

+
+
N/A
+
N/A
+
+
+
+
+

N/A
N/A

N/A

+, Yes; , no; , equivocal; N/A, not applicable.

1426 PART 15 THE GASTROINTESTINAL TRACT

evaluating donor milk samples and alternative human
milk preparations in this context.82 Because most premature infants receive breast milk via the nasogastric route
after artificial collection by mothers and subsequent
freezing, it has been suggested that the lack of the normal
maternal-infant physical interaction during feeding interferes with specific milk immunity, thereby reducing the
protection against the neonates unique microbial flora.
As discussed shortly, the particular microbial profile in
the neonates intestinal environment may contribute to
initiation of NEC.
Specific components of milk feedings have been implicated to cause mucosal injury in the high-risk neonate
and subsequently stimulate the development of NEC.
Studies have shown that hyperosmolar formulae resulted
in disease and that addition of medication to feedings
can markedly increase osmolality.97 Animal studies have
shown that short-chain fatty acids such as propionic or
butyric acid can cause damage to developing intestine
and that colonic fermentation leading to production of
these acids by the host microflora may occur in situations
of carbohydrate malabsorption.12 This pathway may be
especially problematic in the premature infant, partially
deficient in lactase activity and other brush border
enzymes. Finally, an intriguing new hypothesis suggests
that bile acid accumulation may lead to mucosal injury
in the unique environment of the preterm neonate.35
Different approaches to feeding have been associated
with the initiation of NEC. Early studies suggested that
rapid volume increases with full-strength formula
increased the incidence of disease, and protocols were
designed to limit feeding advancement. Several studies
have shown that early hypocaloric or trophic feedings are
safe and improve gastrointestinal function in very low
birth weight (VLBW) infants.83 Feeding advancement has
been evaluated, and the results suggest that judicious
volume increase may be safer,6 although this remains
controversial. It has been postulated that overdistention
of the stomach with aggressive volumes may compromise
splanchnic circulation, leading to intestinal ischemia.
Nonetheless, there remains little clarity on the safety of
differing feeding practices on the incidence of NEC, and
additional trials will be needed to answer this challenging
question.

INTESTINAL ISCHEMIA/ASPHYXIA
Early observations on the pathophysiology of NEC suggested that profound intestinal ischemia led to intestinal
necrosis in unusual clinical situations.90 Similar to the
diving reflex observed in aquatic mammals, it was
hypothesized that in periods of stress, blood flow was
diverted away from the splanchnic circulation resulting
in bowel injury. Although early epidemiologic observations identified asphyxia as an important risk factor, subsequent studies have shown that the majority of NEC
cases are not associated with profound impairment in
intestinal perfusion. In animal models, studies have
shown that the reperfusion following intestinal ischemia
is required in the initiation of bowel necrosis; occlusion
of the mesenteric artery for a prolonged period of time
results in only mild histologic changes atypical for fullblown NEC.

Neonatal animals have been shown to have differences

in the intestinal circulation that may predispose them to
NEC. The basal intestinal vascular resistance is elevated
in the fetus, and soon following birth, decreases significantly, allowing for rapid increase in intestinal blood
flow that is necessary for robust intestinal and somatic
growth.74 It has been shown that this change in the resting
vascular resistance is dependent on the balance between
the dilator (nitric oxide) and constrictor (endothelin)
molecules, and the myogenic response, and altered levels
of these vasoactive mediators have been identified in
human NEC samples.72,73 Perhaps more relevant than
basal vascular tone, studies have shown that the newborn
has alterations in response to circulatory stress, resulting
in compromised intestinal flow and/or vascular resistance. In response to hypotension, newborn animals (3but not 30-day-old swine) appear to have defective
pressure-flow autoregulation, resulting in compromised
intestinal oxygen delivery and tissue oxygenation.71,75 In
addition, in the face of arterial hypoxemia, the newborn
intestinal circulatory response differs from that of older
animals. Although following modest hypoxemia, intestinal vasodilation and increased intestinal perfusion occur;
severe hypoxemia causes vasoconstriction and intestinal
ischemia and/or hypoxia, mediated in part by loss of
nitric oxide production. There are multiple chemical
mediators (nitric oxide, endothelin, substance P, norepinephrine, and angiotensin) that impact on intestinal
vasomotor tone, and in the stressed newborn, abnormal
regulation of these may result in compromised circulatory autoregulation, leading to perpetuation of intestinal
ischemia and tissue necrosis.65,66,76

THE POSSIBLE ROLE OF PACKED RED

BLOOD CELL TRANSFUSIONS IN
NECROTIZING ENTEROCOLITIS
More than 25 years ago, McGrady and colleagues noted
a markedly increased relative risk of developing NEC in
preterm patients who received packed red blood cell
(PRBC) transfusions compared with preterm infants who
did not.61 Subsequent studies have supported the notion
that there is an association between PRBC transfusion
and the development of NEC, particularly with PRBC
transfusions that occur more than a few weeks after
birth, and it has been suggested that these occur temporally within 48 hours after the transfusion has been completed. There are several clinical differences that are
described between PRBC-transfusionassociated NEC
with routine NEC, including PRBC-transfusion NEC
having (1) lower birth weight and gestational age,
(2) more significant anemia, and (3) more varied gestational age, but these have not been universal in all trials.
Based on several case control and cohort studies, there
have now been several reports supporting an association
between PRBC transfusion and neonatal NEC, accounting for 20% to 30% of all NEC cases reported in these
series.21,49 Nonetheless, it remains unclear whether these
transfusions clearly play a causal role in the initiation of
gut injury.
Several mechanisms have been suggested to explain
the phenomenon of PRBC transfusionassociated NEC.

94 Neonatal Necrotizing Enterocolitis

Similar to transfusion-associated lung injury (TRALI) that

is observed in adults following transfusion in a small
minority of patients, La Gamma and others have hypothesized that transfusion-associated gut injury (TRAGI)
occurs in preterm infants, and the pathophysiology of
this response is complex and could involve a variety of
circulatory, immune, and host defense factors that are
activated from stored packed red blood cells and/or PRBC
products.59 A second hypothesis suggests that premature
infants who require transfusion are severely anemic and
the PRBC transfusion results in intestinal blood flow
changes that ultimately activate intestinal necrosis. The
third hypothesis considers that stored PRBC preparations
develop changes that might be risky to the developing
intestine and intestinal microcirculation, including nitric
oxide deficiency, increased bioactive platelet activating
factor (PAF), reduced RBC deformability, and perhaps
increased aggregation, adhesion, and thrombogenic
effects of these banked aliquots. Nonetheless, if PRBC
transfusions do initiate biologic changes leading to NEC,
the etiology is not completely understood.
Because association studies inherently have a high risk
for bias, Kirpalani and Zupancic reviewed the current
understanding of PRBC transfusions and NEC. These
investigators carefully considered the various studies and
found that in the few randomized, controlled PRBC
transfusion trials, there was a higher risk of NEC in the
restrictive, or less transfused group, contradicting most of
the observational studies.47 Nonetheless, meta-analyses
performed for case control studies and cohort studies, as
expected, showed that PRBC transfusions were associated
with a significantly higher risk of NEC. Interpretation of
these opposite effects suggested that a confounder might
independently lead to both transfusion and NEC, and it
may be suggested that the clinical scenario of anemia,
apnea and bradycardia, lethargy, and tachycardia could
lead to the clinical decision to order a PRBC transfusion,
but could also be the classic prodrome for developing
NEC. Therefore, only in a randomized, controlled trial
could these confounders be clearly elucidated, and it is
universally accepted that preplanned randomized, controlled trials are necessary to clarify the clear role of PRBC
transfusions in neonatal NEC.

BACTERIAL COLONIZATION
Although reports have documented isolated epidemics
of NEC associated with specific bacteria (e.g., Clostridia
sp., E. coli, Klebsiella sp., Staphylococcus epidermidis), most
cases occur endemically and demonstrate a variety of
bacterial isolates from stool cultures that are similar to
the flora isolated from patients without intestinal symptomatology.26 Blood cultures are positive in only 20% to
30% of affected cases, and this likely occurs from mucosal
damage and subsequent bacterial translocation. At birth,
the intestine is a sterile environment, and no cases of
NEC have been described in utero, supporting the importance of bacterial colonization in the pathophysiology.
Healthy breastfed infants develop colonization with
several organisms by 1 week of age, including a predominance of anaerobic species of Bifidobacteria and Lactobacilli, whereas the hospitalized, extremely premature infant
intestine has less species diversity and fewer or absent

1427

anaerobes.3,32,41 This imbalance may allow for pathologic

proliferation, binding, and invasiveness of otherwise
nonpathogenic intestinal bacteria, and a reduction in
anti-inflammatory effects and mucosal defense that has
been attributed to probiotic organisms.93 Recent evidence
demonstrates that intestinal bacterial flora are different
in the preterm infant with NEC compared with the
preterm infant without NEC, specifically showing that
NEC patients have less bacterial diversity and more predominance of proteobacterial organisms.58,95 The specific
mechanisms by which bacteria initiate NEC remain
unclear; mounting evidence suggests that bacterial cell
wall products (e.g., endotoxin and lipoteichoic acids)
activate specific toll-like receptors on intestinal epithelium and activate the inflammatory cascade, leading to
the final common pathway of intestinal injury.44,50 Furthermore, accumulating evidence suggests that early
supplementation of probiotic organisms (facultative
anaerobes such as Bifidobacteria and Lactobacilli) reduces
the risk of NEC in animal and human studies; this is
explored further in a subsequent section. In summary,
bacterial colonization is an important factor in the initiation of intestinal injury, and recent evidence suggests that
alterations in the intestinal microbiome may contribute
to the disease. Additional studies investigating the intestinal bacterial transcriptome, proteome, and metabolome
will be necessary to fully understand this very complex
ecosystem.

FINAL COMMON PATHWAY: IMBALANCE

BETWEEN MUCOSAL INJURY AND HOST
DEFENSE/REPAIR WITH ACTIVATION OF
THE PROINFLAMMATORY CASCADE
It has been suggested that mild or moderate stress or
injury to intestinal epithelium (e.g., from feeding, intestinal ischemia, or bacterial products) without adequate
host defense and repair can activate the inflammatory
response leading to intestinal injury and NEC.

Host Defense
Gastrointestinal host defense is markedly impaired in the
preterm infant, and this imbalance further increases the
risk for injury in this population. This intricate system
includes: (1) physical barriers such as skin, mucus membranes, intestinal epithelia and microvilli, epithelial cell
tight junctions, and mucin; (2) immune cells such as
polymorphonuclear leukocytes, macrophages, eosinophils, and lymphocytes; and (3) multiple biochemical
factors.38,92,91 Intestinal permeability to macromolecules,
including immunoglobulins, proteins, and carbohydrates, is known to be greater in the neonate compared
with older children and adults, and in premature infants
this permeability may be more pronounced. Intestinal
mucus, a complex gel consisting of water, electrolytes,
mucins, glycoprotein, immunoglobulins, and glycolipids,
protects against bacterial and toxin invasion, and is
abnormal in developing animals and perhaps premature
infants.85 Additionally, strategic bacteriostatic proteins are
secreted from epithelium that bind to or inactivate the
function of invading organisms. Intestinal trefoil factor is
one such molecule that appears to be developmentally

1428 PART 15 THE GASTROINTESTINAL TRACT

regulated and, therefore, deficient in the premature
neonate.53,88 Human defensins (or cryptidins) are bacteriostatic proteins synthesized and secreted from Paneth
cells that protect against bacterial translocation and are
altered in premature infants and those with NEC.80
Immunologic host defense is abnormal in developing
animals. It is known that intestinal intraepithelial lymphocytes (IEL) are decreased in neonates (B and T cells),
and do not approach adult levels until 3 to 4 weeks of
life. It was recently shown that T-regulatory cells were
decreased in NEC patients, and this could contribute
to excessive inflammation that is characteristic of the
disease.96 Newborns have markedly reduced secretory
IgA in salivary samples, reflecting the decreased activity
presumed in intestine.29 Breast milk feeding provides
significant supplementation; formula-fed neonates have
impaired intestinal humoral immunity, and this deficiency may predispose to the increased incidence of infectious diseases and NEC noted in this population.98
Several biochemical factors that are present in the
intestinal milieu play an important role in the maintenance of gut health and integrity. Substances such as
lactoferrin;60 glutamine;70 growth factors such as EGF,27,28
HB-EGF,30 TGF-,57 IGF,78 and erythropoeitin;45,51 gastric
acid; oligosaccharides;23 polyunsaturated fatty acids;20,54
nucleotides;89 and many others affect mucosal barrier
function, intestinal inflammation, and the viability of
intraluminal bacteria. Many of these factors are deficient
or absent in the preterm neonate, especially in those
patients not receiving breast milk feedings.
Based on a growing body of evidence, mucosal stress
coupled with inadequate host defense and repair can
result in a final common pathway of intestinal injury
involving the activation of the inflammatory cascade.15,16,38
This cascade involves a complex balance of pro- and antiinflammatory endogenous mediators, receptors, signaling pathways, second messengers, and a variety of
downstream effects that ultimately result in end-organ
damage in certain clinical circumstances. Inflammation
can be initiated by a variety of factors; the prototype most
commonly described is by exposure to the bacterial cell
wall product, endotoxin. It has now been clearly shown
that bacterial pathogens initiate downstream events
following the binding of specific pathogen-associated
molecular patterns (PAMPs) to a series of human toll-like
receptors (TLRs) that are expressed on most cells in the
body.1 For example, endotoxin or lipopolysaccharide, the
cell wall product of gram-negative bacteria, binds to and
activates TLR4 that is normally downregulated on the
surface of intestinal epithelium, but has been shown to
be abundantly expressed in stressed animals and human
neonates.44,50 Additional work from these laboratories has
shown that TLR4 dysfunction reduces the risk of experimental NEC in neonatal mice. Following TLR4 activation,
a series of signaling events occurs, allowing for activation
of nuclear factor kappa B and translocation into the
nucleus with subsequent production of a variety of cytokines, including PAF, TNF, IL-1, and IL-8.62,68 In the intestine, early events may include activation of unique cell
death pathways of autophagy and apoptosis,43,69 and subsequent events lead to chemotaxis, transmigration, and
activation of leukocytes, and synthesis and release of

many products from epithelial and inflammatory cells,

such as IL-6, IL-8, IL-10, IL-18, arachidonic acid metabolites, thromboxanes, leukotrienes, and prostaglandins,
nitric oxide, endothelin-1, and oxygen free radicals.31,36,39,40,86,87 If counter-regulatory responses are insufficient (e.g., with decreased or absent IL-1 receptor
antagonist, IL-11, IL-12, PAF-acetylhydrolase, IB leading
to increased nuclear factor kappa B [NFB]), pathologic
changes to gut mucosa occur, and may include accentuated apoptosis of epithelial cells, perturbation of tight
junctional proteins and complexes, increased mucosal
permeability, bacterial translocation, alterations of vascular tone and microcirculation, and additional neutrophil
infiltration and accumulation (Figure 94-2). The process
may then be perpetuated by the activation of the secondary inflammatory response, and the final common
pathway will result in intestinal necrosis. Although these
events remain localized in some cases, in others this activation results in the systemic inflammatory response syndrome, in which patients develop capillary leak,
hypotension, metabolic acidosis, thrombocytopenia,
renal failure, respiratory failure, and often, death. In
summary, proinflammatory signaling follows TLR4 activation on the intestinal epithelium and leads to a cascade
culminating in intestinal necrosis in neonatal animals,
similar to human, neonatal NEC.
Although endotoxin is a well-characterized activator of
inflammation, additional factors may play a role in stimulating the NEC cascade in premature infants. Asphyxia
and/or ischemia-reperfusion activate the early mediators
of inflammation in many tissues, including intestine.
Neonatal animal studies have shown that the stress of
formula feeding stimulates phospholipase A2 gene expression, intestinal PAF production, and stimulation of apoptosis and the inflammatory response with resulting
NEC.15 Therefore, many of the purported risk factors
for NEC may activate the inflammatory response that
results in the final common pathway described in the
preceding.
The evidence suggests that the premature neonate may
have an abnormal balance between pro- and antiinflammatory mediator regulation, thereby increasing
their predisposition for diseases such as NEC. PAF is a
potent phospholipid inflammatory mediator that is associated with NEC in several experimental models and
human analyses.18,19,33 PAF infusion causes intestinal
necrosis in animals, and PAF receptor antagonists prevent
injury following hypoxia, endotoxin challenge, TNF
infusion, and ischemia-reperfusion. It has been shown
that neonates are markedly deficient in their ability to
degrade PAF, owing to decreased activity of the PAFspecific enzyme PAF-acetylhydrolase.19 Of interest, PAFacetylhydrolase knockout mice have a much higher
incidence of NEC compared with control mice, supporting the importance of anti-inflammatory protection in
this model.55 PAF-acetylhydrolase is present in breast
milk but absent in commercial formula, and this may in
part explain the beneficial effects of breast milk feeding.
Interleukin-10 is an anti-inflammatory cytokine thought
to be important in reducing intestinal inflammation and
possibly NEC in animals and humans. In neonatal rats,
maternal milk feedings increased IL-10 and reduced the

94 Neonatal Necrotizing Enterocolitis

Intestinal
ischemia

Formula
feeding

Bacteria

1429

Prematurity

TLRs

Mucosal injury

Host defense

Proinflammatory mediators

Anti-inflammatory mediators

IL-1, 6, 8, 18
PAF, ET-1, leukotrienes, thromboxanes
Free oxygen radicals
NFB

IL-1 RA, IL-11, 12

PAF-acetylhydrolase
IB
Growth factors, e.g., EGF, EPO, IGF

Altered microcirculation

Figure 94-2 Hypothetical events in the pathophysiology of neonatal necrotizing enterocolitis

(NEC). EGF, Epidermal growth factor; EPO, erythropoietin; ET, endothelin; IGF, insulin-like growth factor;
IL, interleukin; PAF, platelet activating factor; TLR, tolllike receptors; NFB, nuclear factor kappa B.

incidence of NEC, whereas in human milk specimens, a

significant percentage of NEC patient-pairs were deficient
in this important cytokine. Studies have compared proinflammatory response to endotoxin and/or IL-1 in different cell lines, and have found that IL-8 response is
significantly higher in fetal intestinal epithelium compared to mature, adult intestine.22,68 Finally, it was shown
that nuclear factor B (NFB) activation is poorly downregulated in neonatal animals, in part because of immaturity of IB.24 These results suggest that the neonatal
balance of the inflammatory response may be weighted
toward the proinflammatory side and more likely to
result in the pathologic outcome of NEC.

Prevention of Necrotizing
Enterocolitis
Based on the unique epidemiologic features and understanding of the pathophysiology, there have been multiple approaches attempted to prevent NEC in animal and
human studies. It should be appreciated that human prevention trials with sufficient power to demonstrate a
reduction in NEC incidence from 10% to 5% (e.g., in
babies born weighing <1500g) would require a large
number of patients, approximating 350 patients per treatment group. Reduction of disease in animal models has
been shown with breast milk feeding, IgA supplementation, antibiotic prophylaxis, steroids, probiotics, polyunsaturated fatty acids, platelet-activating-factor antagonists,
PAF-acetylhydrolase, epidermal growth factor, TGF-,

Apoptosis

Mucosal permeability

Inflammation

NEC

trefoil factor, leukocyte depletion, and oxygen radical

scavengers. In human studies, there remains no standard,
effective alternative for NEC prevention, although careful
enteral feeding with breast milk is the best approach
neonatologists have to offer. Prevention trials with IgA/
IgG,29 steroids,34 polyunsaturated fatty acids,20 arginine,2
and antibiotics84 have been conducted with limited
success, but because of various problems, including poor
study design, risks of intervention, lack of reproducibility,
and weak statistical power, these approaches have yet to
become routine strategies in the neonatal intensive care
unit for preterm infants.
Probiotic supplementation is a promising approach
for the prevention of NEC in very low birth weight
(VLBW) infants. There have now been more than 20 international prospective, randomized trials that demonstrate
efficacy for probiotic prophylaxis for this indication, and
additional US trials are planned to confirm these findings
(Table 94-3; and see Wang etal., 2012 for recent metaanalysis).94 As described, probiotic colonization in VLBW
infants appears inadequate, and studies have defined
multiple plausible mechanisms whereby probiotics could
improve gastrointestinal health and prevent proinflammatory signaling and disease. As seen in Table 94-3, there
is a significant risk reduction in NEC (RR = .36, p < .01)
and mortality (RR = .58, p < .01) with probiotic supplementation, although the specific probiotic species and
dosing varied somewhat among trials, and NEC was not
the primary outcome of interest in many of these studies.
Nonetheless, the cumulative results identified a reduction
in NEC from 157/2328 controls (6.7%) to 58/2388

1430 PART 15 THE GASTROINTESTINAL TRACT

TABLE 94-3

Probiotic Trials for Neonatal Necrotizing Enterocolitis

Study or
Subgroup

Probiotics
Events

1.1.1 All Infants

Bin-Nun, 2005
1
Costalos, 2003
5
Dani, 2002
4
Kitajima, 1997
0
Lin, 2005
2
Lin, 2008
4
Manzoni, 2006
1
Manzoni, 2009
0
Mohan, 2006
2
Roug, 2009
2
Samanta, 2009
5
Sari, 2010
6
Stratiki, 2007
0
Subtotal (95% Cl)
Total events
32
Heterogeneity: Chi2 = 11.08, df = 11
Test for overall effect: Z = 5.20 (P <

Control

Total

Events

Total

Weight

72
51
295
45
180
217
39
151
37
45
91
110
38
1371

10
6
8
0
10
14
3
10
1
1
15
9
3

73
36
290
46
187
217
41
168
32
49
95
111
31
1376

10.9%
7.7%
8.8%
10.8%
15.3%
3.2%
10.9%
1.2%
1.0%
16.1%
9.8%
4.2%
100.0%

Risk Ratio

Risk Ratio

M-H, Fixed, 95% Cl

M-H, Fixed, 95% Cl

0.10 [0.01, 0.77]

0.59 [0.19, 1.78]
0.49 [0.15, 1.61]
Not estimable
0.21 [0.05, 0.94]
0.29 [0.10, 0.85]
0.35 [0.04, 3.23]
0.05 [0.00, 0.90]
1.73 [0.16, 18.20]
2.18 [0.20, 23.21]
0.35 [0.13, 0.92]
0.67 [0.25, 1.83]
0.12 [0.01, 2.19]
0.35 [0.24, 0.52]

90
(P = 0.44); I2 = 1%
0.00001)

0.01
0.1
1
10
100
Favors treatment
Favors control

Effect of probiotic prophylaxis on severe neonatal NEC (stages II-III). These studies all randomized premature infants (with variable gestational-age/birth weight
cutoffs) to probiotic (variable doses and schedule) and placebo, and reported on the incidence of NEC in the cohorts.
From Alfaleh K, Anabrees J, Bassler D, Al-Kharfi T. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2011;(3):CD005496.

treated (2.4%, p < .01). Of additional importance, there

were no reported cases of probiotic sepsis in any of these
cohorts, although it should be noted that routine anaerobic culture techniques were routinely not performed.
Despite this uncertainty, mortality rates overall were
lower in patients treated with probiotics (96/1941, 4.9%,
RR = .56, p < .01) compared to controls (166/1962,
8.4%).
Although there is much excitement and appropriate
optimism regarding this promising strategy, there are
several important reasons why additional studies are warranted before a clear standard of care can be recommended. First, different probiotic species have differing
effects, and the optimal probiotic combination and
optimal dosing strategy are not clearly elucidated. Second,
probiotic preparations have not been rigorously regulated, and because some studies have shown inaccuracies
in the reported organism species and content, appropriate quality control measures are warranted.25 Finally, probiotic sepsis has been observed in this unique population,42
and additional safety concerns have been raised in recent
reports, demonstrating increased death in an adult population of ICU patients, and increased wheezing and
asthma in pediatric patients treated in the newborn
period.7,46 If a multicenter trial performed in the United
States demonstrates efficacy similar to that reported in
other countries, probiotic use may become routine in US
NICUs.

Summary
In conclusion, NEC is a clinical burden to patients, families, and the neonatology health care team. Although the
diagnosis is straightforward, the morbidity and mortality

associated with the disease is not improving. Risk factors

of prematurity, formula feeding, intestinal ischemia/
hypoxia, and bacterial colonization accentuate the imbalance toward mucosal stress with impaired host defense,
in some cases leading to uncontrolled intestinal inflammation and necrosis. The premature infant differs from
term infants and older patients in multiple ways, including enteral feeding characteristics, bacterial colonization
patterns, autoregulation of splanchnic blood flow, host
defense, and the regulation of the inflammatory cascade.
Although several strategies to prevent NEC have been
tested in humans and animals, most have enjoyed limited
success. Perhaps the simplest and safest intervention is to
increase the use of human milk in this population. Probiotic supplementation studies have been exciting, and
following additional investigation, their use may significantly impact the incidence, morbidity, and mortality
associated with neonatal NEC.

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