67

SYMPOSIUM : NEONATOLOGY II
Evidence and Experience in Neonatal Medicine
Necrotising Enterocolitis: The State of the Science
KathleenGibbs,JingLinandIanR.Holzman
Division of Newborn Medicine, Department of Pediatrics, The Mount Sinai School of Medicine, New York, NY
ABSTRACT
Necrotizing enterocolitis is the most common gastrointestinal emergency of the neonate, affecting 5-10% of infants, yet the
pathogenesis remains unclear. Widely accepted risk factors include prematurity, enteral feeds, bacterial colonization and
mucosal injury. How these or other yet identified factors come together to create the classic clinical and pathologic features
is the subject of much research. The activation of the cytokine cascade, in part by bacterial ligands, appears to play a key role
in mucosal injury. Two mediators that may also contribute are platelet activating factor and intestinal toll-like receptors. Short
chain fatty acids, the products of bacterial fermentation of carbohydrates, have been thought to cause mucosal injury.
Overgrowth of pathogenic bacteria in the face of a decreased commensal population may play a key role. A current focus of
clinical research involves probiotics, enterally fed forms of commsenal bacteria. This may set the stage for a healthier intestinal
ecosystem and possibly, decreased risk of NEC. [Indian J Pediatr 2007; 74 (1) : 67-72] E-mail: Ian.Holzman@MSSM.EDU
Key words : Necrotizing enterocolitis; Platelet activating factor; Toll-like receptor, Short chain fatty acids, Probiotics
NecrotizingEnterocolitis(NEC)isthemostcommon
gastrointestinalemergencyoftheneonate,yetthetrue
etiologyremainsuncleardespiteyearsofresearch. Many
theorieshaveattemptedtoelucidatethetruepathogenesis
overthelast40yearswithoutsuccess. Thesetheoriesand
subsequentresearcheffortshavecenteredonwhatarefelt
tobethemostimportantriskfactors:prematurity,enteral
feeds, mucosal injury and the presence of bacteria.
Historically,clinicalresearchhasfocusedparticularlyon
feedingpracticeswhen,withwhatandhowfastto
increase them. Attempts to identify a consistent
infectiousetiologyhavefailed,yetstrictinfectiouscontrol
parameters can decrease the incidence. With the
advancesofmolecularbiology,theidentificationofafew
factorsholdspromise,includingplateletactivatingfactor
(PAF),intestinaltoll-likereceptors(TLR),shortchainfatty
acids(SCFAs),andtheactivationofthecytokineinduced
inflammatorycascade. Theyappeartoplayapivotalrole
innotonlymucosalinjurybutthemaintenanceofan
intactmucosalbarrier. Oneareaofclinicalresearchis
focusedonprobioticsandtheirimpactonthenormal
commensalflora. Thisarticlewillreviewthemostcurrent
theoriesonthepathogenesisofNEC,allwhichrevolve
aroundthemostwidelyacceptedriskfactors.
Prematurity
Prematurityremainsthemostconsistentfactor,although
CorrespondenceandReprintrequests:Dr.IanR.Holzman,Chief,
DivisionofNewbornMedicine,TheMountSinaiSchoolofMedicine,
Box1508,OneGustaveL.LevyPlace,NewYork,NY10029.
termbabiescandevelopNEC.Approximately5-10%of
infantswithabirthweightlessthan1500gramswill
developNECandtheincidenceincreaseswithdecreasing
gestationalage.
1,2
Inthe1960sSantullidescribedsomeof
thefirstcasesofNECinprematureinfantswithlowapgar
scoresandhyalinemembranedisease. Theseinfantswere
dependentuponventilatorysupport,hadumbilical
cathetersinplace,anddevelopedthehallmarksignsof
NECaftertheinitiationofenteralfeeds.
3
Thesepatients
providedthebasisfortheconceptthatinorderforNECto
occur,threethingsneededtobepresentbacteria,
ischemia,andsubstrate(enteralfeeds). Neonatologistsin
the modern NICU find themselves with far fewer
asphyxiated preterm infants, more limited use of
umbilicalcatheters,shortertimeonventilators,and
judiciousfeeding.
Asaresult,thefocushasshiftedtotheroleofthe
immaturemucosalbarriercoupledwithanimpaired
immune response to intestinal pathogens. While
secretory IgA (sIgA) doesnt participate in the
inflammatory cascade its deficiency may facilitate
bacterialtranslocation.
4
ItisknownthatsIgAplaysan
importantroleinmucosaldefensebybindingwith
antigensandimpairingtheirabsorptionbyenterocytes.
Asecondsuggestedimmunesysteminteractionisthe
possibilitythataberrantTlymphocyteactivitymayfailto
recognize a breakdown in the mucosal barrier in a
prematureinfant.
4
Feeding
Enteralfeedshavealmostalwaysbeenadministered
whenNECoccurs,althoughsymptomsusuallydevelop
Indian Journal of Pediatrics, Volume 74January, 2007 67
68
Kathleen Gibbs et al
weeksaftertheintroductionoffeeds,butoftennotfar
afterachievingfullenteralfeeds. Manytimesinfants
develop symptoms following recent volume
advancementorafterreinitiatingfeeds. Humanmilk
reducestheincidencebutdoesntpreventitentirely.
Barlowpublishedastudyin1974citingtheimportanceof
humanmilkindecreasingtheriskofNECandsuggesting
theroleofprotectivefactorssuchassIgAmentioned
above.
5
Others have speculated on the effects of
osmolalityindamagingtheintestinebutpublisheddata
havefailedtosupportthis. Whilemanyclinicianshave
writtenonthetopicofsafefeedingadvancement,the
availablestudiesareconflicting. Mostauthorsagreethat
20ml/Kg/dayisasafeadvancementrate.
6
ManyNICUs
haveapolicyofattemptingminimalenteralnutritionor
trophicfeedsforaperiodoftimebeforeadvancingfeeds
inanattempttoprimetheintestine.
InfectiousAgents
Itisclearthatinfectiousagentsplayaroleintheclinical
appearance of NEC. No single pathogen has been
identifiedgramnegativebacteriaarethemostcommon,
followedbygrampositivebacteria,butyeastandeven
viruseshavebeenimplicated. CasesofNECareusually
sporadic, but the many reports of clusters suggest
colonizationwithparticularlyvirulentstrainsmaybe
important. Only1/3ofinfantswillhaveapositiveblood
culture,butbacteremiaisseenmoreoftenwithadvanced
disease. Pneumatosisintestinalis,thehallmarkradiologic
findingofNEC, istheintraluminalandportalsystem
presenceofhydrogengasproducedbythefermentation
ofcarbohydratesbybacteria.
Thegastrointestinaltractofthenewborninfantis
sterilebutcolonizedwithin12-24hoursfirstwith
maternalvaginalflora,followedbythatoftheexternal
environment.ThenormalcommensalfloraLactobacilli
andBifidobacteriumarefoundweekslater. Colonizationis
affectedinpartbyfeedingpractices.Feedingwithhuman
milkisassociatedwithearliercolonizationbyanerobic
commensals.ThetherapeuticstrategiesusedintheNICU
broadspectrumantibioticsanddelayedinitiationof
enteralfeedscontributestothefactthattheVLBWinfant
often has a delayed onset and aberrant pattern of
colonization.
7
Thismayfavorthepresenceofmore
pathogenicstrainsovertheusualcommsensalflora.
Ischemia
IschemiahaslongbeenthoughttoplayaroleinNEC. It
wasthoughtthatischemiawastheincitingevent,with
interruptedmucosalintegrityandbacterialtranslocation
theendresult.Thehallmarkhistologicalfindingsof
mucosal edema, ulceration, inflammation and
coagulationnecrosissupporttheacceptedbeliefthat
ischemiaplaysaroleatsomepoint.Historicalriskfactors
suchasperinatalasphyxia,presenceofumbilicallines,
polycythemia,hypotension,ortheuseofindomethacin
supportedtheroleofischemia. However,epidemiologic
studieshavefailedtoconfirmanassociationbetween
NECandmostoftheseriskfactors.
Currentresearcheffortsarefocusedontheroleof
intrinsicvascularregulation.Theintestinalcirculationof
the newborn has a low vascular resistance which
facilitatesahighrateofbloodflow.Thisregulationoccurs
bythebalancingofelementsthatfavordilatationor
constrictionofthevasculature.Endothelin-1isapeptide
thatpromotesvasoconstriction.Nitricoxide(NO)isafree
radicalproducedbyanintactendotheliumthatfavors
vasodilatation.NOproductionoccursbywayofan
enzyme-endothelialnitricoxidesynthetaseoreNOS-
expressedatlowlevelsduringfetallifeandincreased
postnatally.Atbaselinethebalancefavorsanexcessof
NOandthusavasodilatedintestinalmicrocirculation.
Theprincipalsitesofresistancearethearterioleswithin
thesubmucosalplexus.
8
Eventsthatalterthisbalance
wouldshifttheequilibriumtowardsincreasedvascular
resistanceandthusalimitationofbloodflowandoxygen
delivery.
8
Ischemia-reperfusioninjuryfollowingperinatal
asphyxia,ortheprolongedstateoflowflowperfusionin
growthretardedfetusesaretwoexamplesthatareafocus
ofconsiderableresearchinterest.Animalmodelssupport
theirroleinthedysregulationofintestinalvascular
resistance,primarilybydisruptingNOproduction.
8
However,whethertheimplicatedroleofischemiaisthe
incitingcauseforNECortheendresultofaninitial
inflammatorydisruptionofthemucosalbarrierremains
unknown.
InflammatoryMediators
Current evidence points to the role of activated
inflammatory mediators and an inadequate anti-
inflammatoryresponseinthebreakdownofmucosal
integrity, which may represent the final common
pathwayinNEC. Inclinicalstudies,patientswithNEC
often have elevated inflammatory mediators and
attemptshavebeenmadetocorrelatethepresenceand
concentrationofthesefactorswithdiseasepresentation.
9
Intestinal epithelial cells produce many of the
cytokinesthatareimplicatedasmediatorsofintestinal
inflammationandinjury.Cytokinesaresolublemolecules
thatbindcellreceptorsandplayaroleinbothhumoral
andcell-mediatedimmuneresponses.IL-6induces
secretion of acute phase proteins, enhances T cell
proliferationandantibodyproductionbyBcells.IL-8
recruitsinflammatorycells.ThesynthesisofTNFis
generated in part by microbial products and other
cytokines. TNF recruitsinflammatorycells,butisalso
involvedincytotoxicityandprogrammedcelldeath.
PlateletActivatingFactor(PAF)isoneofthemediators
most intensely studied. PAF is an endogenous
phospholipidinflammatorymediatorthatisproducedby
inflammatory cells, endothelial cells, platelets and
bacteria.TherearePAFreceptorsonmostcells.The
Indian Journal of Pediatrics, Volume 74January, 2007 68
69
Necrotising Enterocolitis : The State of the Science
receptorgeneisexpressedinmanyorgans,butevidence
existsthatthegreatestreceptorexpressionisfoundinthe
ileum-themostcommonsiteofinvolvementinNEC.
10
ActivationofthePAFreceptorinducestheproductionof
additionalmoleculessuchasTNF,IL-6,andIL-8.In
addition,itactivatespathwaystriggeringapoptosisin
intestinalepithelialcells.
10
Invariousexperimental
models, PAF causes capillary leak, myocardial
dysfunction, renal dysfunction, neutropenia,
thrombocytopenia,andhypotension.
10
Conversionby
PAF-acetylhydrolaserendersitinactive. Humanneonates
havelow/absentcirculatingPAF-acetylhydrolase,and
humanmilkcontainssignificantquantities.
Animalmodelshaveattemptedtoreproducethe
clinicalandpathologicfeaturesbasedonacceptedrisk
factors. Barlowcreatedthefirstmodelinthe1970sof
newbornratsstressedwithasphyxia,colonizedwith
entericbacteriabynasogastrictubes,andsubsequently
fed. Itwasthisanimalmodelthatfirsthighlightedthe
importanceofbreastmilkinpreventingNEC.
5
More
recently,investigatorshaveusedasimilaranimalmodel
toillustratetheimportanceofendogenousmediatorssuch
asPAFinthepathogenesisofNEC.Theyinducedbowel
necrosisbyinfusionofPAF,TNForlipopolysaccharide
(LPS). PAFinfusioncausedfocalnecrosis,butaffected
theentiresmallbowelwhengiveninlargequantities.
LPSactedasaprimingagentforPAF,assmalldoseswere
synergisticwithlowdosesofPAF. LPS-inducedintestinal
injury was blocked by pretreatment with a PAF
antagonist.
11
Toll-LikeReceptors
Toll-likereceptors(TLRs)onthecellsurfaceactassensors
ofmicrobialinfectionandplayaroleintheinitiationof
theinflammatoryandimmunedefenseresponse. Both
commensalandpathogenicbacteriasecretemolecules
thatserveasligandstoTLRs,suchaslipopolysaccharide
(LPS)andlipoteichoicacid(LTA). LPShasbeenshownto
actspecificallyonTLR4,andLTAonTLR2. TLRsmay
playaroleinthepreservationofintestinalepithelial
integrity.
Rakoff-Nahoumandcolleaguesstudiedtheinteraction
of TLRs and commsensal organsisms in a series of
experimentswithmice.
12
Theyfoundthatactivationof
TLRsbythecommsensalfloraplayedafundamentalrole
not only in the preservation of intestinal epithelial
integritybutalsoinprotectionfrominjury.Animals
deficientinTLRsignalingweremorevulnerableto
inducedintestinalinjuryascomparedwithcontrols,
resultinginanear100%mortalityasopposedto100%
survival. Inaddition,theseanimalsproducedlowlevels
offactorsintegralincellularprotectionandrepairbefore
andafterinjury. Aninterestingcomponentoftheseseries
ofexperimentswastheroleofcommensalsinprotection
frominjury.Wildtypemicedepletedofcommensalsby
anantibioticregimenhadsimilarratesofmorbidity/
mortalityfollowinginducedintestinalinjuryasdidthe
micedeficientinTLRsignaling,andthesemicehadlow
concentrationsofthefactorsthatplayakeyroleincellular
protectionandrepair. TheauthorsconcludedthatTLRs
arecrucialinmaintainingintestinalepithelialhomeostasis
particularlyfollowinginjury,andthatcommensalflora
inducetheproductionofprotectivefactorsfollowing
epithelialinjury.
However,activationofTLRsdoesresultincytokine
activationand,potentially,aconsiderableinflammatory
response.Therefore,especiallyinaneonatelacking
commensalflora,TLRsmayplayaroleintheactivationof
apathologicinflammatoryresponseandsubsequent
injury.ColonizationwithorganismsthatsecreteLPSor
LTA,knownligandsforTLRs,occursearlyandpriorto
thatofanaerobiccommensals.Caplanet al.proposeda
potentialroleofTLRsinactivationofthepathologic
inflammatoryresponse,asopposedtoprotectionfrom
injury.
10
InanewbornmicemodelofNEC,asphyxiated
animals fed with formula were found to have up-
regulatedTLR4expression. Inaddition,PAFgiventothe
animalsinducedTLR4expressionandTLR4deficient
micehadadecreasedincidenceofNEC.Theauthors
concludedthatactivationofTLR4bybacterialligandsand
PAF may be critical in the initiation of events that
culminateinNEC.
ShortChainFattyAcidsandBacterialOvergrowth
Itiswellacceptedthatthepresenceofbacteriausuallyis
aprerequisiteforNECtodevelop,yettheactualroleof
bacteriaisundefined. Dotheyincitetheinjuryordothey
gainaccesstothecellsbelowtheepithelialliningonly
afterinjuryoccursbyanothermechanism? SCFAs,
mainlyaceticacid,propionicacidandbutyricacid,arethe
productsofbacterialfermentationofcarbohydratesinthe
intestinallumen. Innewborninfants,normalintestinal
bacterialcolonizationisestablishedonceenteralfeedingis
achieved.Intheanaerobicenvironmentofthecolon,
bacteria rapidly ferment carbohydrates to gases
(hydrogen,carbondioxide,andinsomecases,methane)
andSCFAs.
13
Thisprocessofbacterialfermentationof
undigestedcarbohydratesplaysanimportantrolein
normal intestinal biology such as water and salt
absorptioninthecolon,energysalvation,andcolonic
mucosalmaturation.
13
Intheprematureinfantwhohasa
relativelactasedeficiency,lactoseingestedintheformof
milkmaybefermentedintoSCFAsandsubsequently
absorbed.Insomeprematureinfants,anabnormalstateof
SCFAover-productionmayariseduringperiodsof
significantcarbohydratemalabsorptionand/orbacterial
overgrowth. This may exceed the buffering and
absorptivecapacityofthecolonandleadtoanincreased
concentrationofSCFAsinthecolon.
13
LevelsofSCFAsin
thedistalileummayalsoincreasebywayofrefluxacross
theileo-cecalvalveorduetolocalbacterialovergrowth.
Lin et al have demonstrated that intraluminal
Indian Journal of Pediatrics, Volume 74January, 2007 69
70
Kathleen Gibbs et al
administrationofSCFAscaninduceconcentrationand
maturation-dependent intestinal mucosal injury in
newbornratswithpathologysimilartothatseenin
NEC.
14,15
Intraluminaladministrationoflacticacid,the
fermentationproductoflacticacid-producingprobiotics,
doesnotinduceidentifiableintestinalmucosalinjury.
Theirfindingssuggestthatoverproduction/accumulation
ofSCFAs,butnotlacticacid,intheproximalcolonand/
ordistalileummayplayakeyroleinthepathogenesisof
NEC.
ArecentlypublishedstudybySangildet aluseda
novel design in an animal model to illustrate how
formulafeedingandbacterialcolonizationcouldinduce
NECwithoutapriorasphyxialinsult.
16
Inaseriesof
experiments, they documented the spontaneous
Aberrantcolonization
Overgrowthofpathogenicbacteria
LTA LPS
TLR??
developmentonNECinformulaandcolostrumfed
prematurepigs.Animalsfedformulahadhigherratesof
NEC(53%vs5%inthecolostrumgroup),associatedwith
villousatrophyeveninthosethatappearedclinically
healthy.Animalsfedcolostrumhadhigherlevelsofbrush
borderenzymes. Animalsraisedinasterileenvironment
andsubsequentlyfedlackedvillousatrophy,andformula
fedanimalswerefoundtohavehighratesofClostridium
colonization. Afterformulafedanimalswerepassively
immunizedwithplasmacontaininghighlevelsoftiters
againsttoxinstoC.perfringensandE.coli,theyfailedto
developclinicalorhistopathologicsignsofNEC.This
studyimplicatestheinteractionofbacteriaandsubstrate
inthepathogenesisofNEC.
Probiotics
Breastmilk
Commensalflora
PAF
PAF-AH
Formulafeeding
SCFA
Ischemia
Activationofthecytokinecascade
MucosalInjury
BacterialTranslocation
NEC
Fig1. Flowdiagramofproposedfactorsinvolvedinthepathogenesisofnecrotizingenterocolitis.LTA-Lipoteichoicacid;LPS
Lipopolysaccharide;TLRToll-likereceptor;PAFPlateletactivatingfactor;PAF-AHPlateletactivatingfactoracetyl-hydrolase;
SCFAShortchainedfattyacids;NECNecrotisingEnterocolitis.
Indian Journal of Pediatrics, Volume 74January, 2007 70
71
Necrotising Enterocolitis : The State of the Science
TABLE.Probiotics:RecentClinicalTrials
Author StudyMethod Subjects Probiotics Primaryoutcome Finding
Dani ProspectiveRCT N=585total Dicoflor UTI Notsignificant
Italy,2002 12NICUs <33wks,<1500g Lactobacillus GG Bacterialsepsis
NEC
BinNun ProspectiveRCT N=145total
Israel,2005 1NICU <1500g ABCDophilus NEC Significant:Cases
Bifidobacteriainfantis and/or andseverityof
Streptococcusthermophilus Death NEC
Bifidobacteria bifidus
NotSignificant:
NECassociated
mortality
Lin ProspectiveRCT N=367total Infloran NEC Significant:Reduction
Taiwan,2005 1NICU <1500g Lactobacillus acidophilus or inincidenceand
Bifidobacterium infantis Death severityofNEC
Probiotics
Ithasbeenincreasinglyrecognizedthatnormalhealthy
gutmicrofloraplayavitalroleinhumanhealthand
performimportantmetabolicfunctionsthatsupportthe
digestivesystem.Probioticsarelivingmicroorganisms,
whichuponingestionincertainnumbersexerthealth
benefits beyond inherent general nutrition. Most
probioticsarelacticacid-producingbacteria.Thisisa
largegroupofbacteriasonamedbecausetheyproduce
lacticacidasanendproductoffermentation.Probiotics
currentlybeinginvestigatedinclinicalpracticeare
enterallyfedformsofthesenormalcommensalsthatfail
totranslocateorcausemucosalinjuryandinfactmay
protect against mucosal injury. Among them,
BifidobacteriumandLactobacillusaresomeofthemost
commonprobioticsfoundintheintestineofhealthy
newborninfants
Administrationofsomecommonprobioticshasbeen
showntoreducetheincidenceofNECinexperimental
animalmodelsaswellasinclinicaltrials.Usingananimal
modelofNEC,Caplanetaldemonstratedthatprobiotics
mayplayaroleinthepreventionofNEC.
17
Ratswere
inoculatedwithB. infantis,E.coli,orsalinecontroland
exposed to an NEC protocol (formula feeding +
asphyxia).TheydemonstratedcolonizationwithB.
infantisintheintestineandstoolwithin48hours. Levels
oftheinflammatorymediators,plasmaendotoxinand
phospholipaseA2,werelowerintheB. infantisgroup. In
addition,B. infantistreatedanimalshadasignificant
reductionintheincidenceofNECcomparedwithcontrol
andE.colitreatedanimals.
Theuseofprobioticsintheclinicalsettinghasbeena
sourceofgreatinterest,andafewpublishedreports
supporttheiruse.Threerecentprospectiverandomized
trialshavesoughttoillustratethebeneficialimpactof
enterallyfedprobioticsinthepreventionofNEC (Table).
Intwoofthestudies,inwhichtheprimaryoutcomewas
death or significant NEC, a statistically significant
reduction in the incidence and/or severity of NEC
occurredintheprobioticfedgroup.Bothofthesecenters
hadafairlyhigh(15and23%)pre-studyincidenceof
NEC.
18,19
Thethirdstudy,whichexaminedprobioticuse
inthepreventionofurinarytractinfections,bacterial
sepsisandNEC,foundnostatisticallysignificantimpact.
20
Insummary,thetypicalpatientwithNECisatits
simplestaprematureinfantwhohasbeenfed. Whatsets
thestageforthispotentiallydevastatingillnessremains
oneofthegreatmysteriesofneonatology.Oncethe
mucosalbarrierisdisruptedbacterialinvasionintothe
walloftheintestinecouldoccur. Bacterialtranslocation
maypropagatefurtherinjurybytheadditionalactivation
ofinflammatorymediators.Theendresult,ischemia,
apoptosis,andconcomitant mucosaldisruptionleadsto
pneumatosisandthecharacteristiclesionsofcoagulation
necrosis. InflammatorymediatorssuchasTNFandIL-
8arewellknowntoinduceshockandthesystemic
inflammatoryresponsesyndrome(SIRS). Ifthisvicious
cyclecontinues,thereisahighlikelihoodofintestinal
necrosisandperforation.Usingprobioticstoproducea
healthierintestinalecosysteminprematureinfantsmay
haveapotentialroleinthepreventionofNEC,anelusive
goalfordecades.Nolongermustwesacrificeearly
enteralnutritionalsupport(anapproachthatcanleadto
fewercentrallinesinfectionsandlesshepatotoxicityfrom
parenteralnutrition)toourconcernsabouteventual
bowelnecrosis. Whilethestorywillcontinue,theending
isnowatleastinsight!
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