NF-kappaB-mediated expression of MAPK phosphatase-1 is an early step in desensitization to TLR ligands in enterocytes.

Toll-like receptor (TLR) signaling in naive enterocytes is rapidly inhibited, leading to the establishment of tolerance. To gain insight into tolerance at the level of the proinflammatory mitogen-activated protein kinase (MAPK) p38, we characterized TLR-mediated induction of the p38-specific phosphatase MKP-1. In cultured enterocytes, ligands of TLR3, TLR4, TLR5, and TLR9, but not TLR2, induce MKP-1 at 30-60 min, coincident with dephosphorylation of p38 following the peak of TLR ligand-induced phosphorylation. Induction of MKP-1 is blocked by inhibitors of nuclear factor (NF)-kappaB, but not of MAPK. Small interfering RNA knockdown of IkBalpha prolongs the expression of MKP-1. Rat MKP-1 promoter contains two NF-kappaB-binding sites, mutations in which additively impair lipopolysaccharide-induced transcription from the MKP-1 promoter. In the intestine, MKP-1 is expressed in the crypts, the epithelial compartment that also displays bacteria-dependent activating phosphorylation of p38. Thus, NF-kappaB-dependent expression of MKP-1 may contribute, by desensitization of p38, to the rapid establishment of unresponsiveness to several TLR ligands in enterocytes.

Evidence vs experience in the surgical management of necrotizing enterocolitis and focal intestinal perforation.

INTRODUCTION: Necrotizing enterocolitis (NEC) and focal intestinal perforation (FIP) are neonatal intestinal emergencies that affect premature infants. Although most cases of early NEC can be successfully managed with medical therapy, prompt surgical intervention is often required for advanced or perforated NEC and FIP. METHODS: The surgical management and treatment of FIP and NEC are discussed on the basis of literature review and our personal experience. RESULTS: Surgical options are diverse, and include peritoneal drainage, laparotomy with diverting ostomy alone, laparotomy with intestinal resection and primary anastomosis or stoma creation, with or without second-look procedures. CONCLUSIONS: The optimal surgical therapy for FIP and NEC begins with prompt diagnosis and adequate fluid resuscitation. It appears that there is no significant difference in patient outcome based on surgical management alone. However, the infant's weight, comorbidities, surgeon preference and timing of intervention should be taken into account before operative intervention.

Evidence vs experience in neonatal practices in necrotizing enterocolitis.

INTRODUCTION: Necrotizing enterocolitis (NEC) has been recognized for over 40 years as a cause of inflammation and necrosis of the small and large intestine of infants born at less than 36 weeks of gestation. NEC remains a significant health problem for infants born prematurely and may become the leading cause of morbidity and mortality among these infants worldwide. The sequence of events leading to NEC is complex and multifactorial, although damage to the intestinal epithelium and invasion by bacteria are known to play central roles in disease pathogenesis. STUDY DESIGN: Bacteria initiate a cascade of inflammation that may progress to intestinal necrosis and perforation with sepsis and death. RESULT: Treatment of infants at risk for NEC with probiotic bacteria may be an area of great potential, as probiotic bacteria may promote maturation of the epithelial barrier and function to exclude bacterial pathogens from critical niches in the intestine, thereby disrupting a primary pathway in disease pathogenesis. CONCLUSION: Understanding how probiotic bacteria, or other novel therapies, prevent or limit disease propagation in NEC will be paramount in limiting the impact of disease in a growing population of premature newborns.

Improved functional outcome for severely injured children treated at pediatric trauma centers.

BACKGROUND: Controversy exists regarding the impact of pediatric trauma centers (PTC) on survival for injured children. However, functional outcome for children treated at PTC compared with adult trauma centers (ATC) has not been evaluated. METHODS: An analysis of children entered in the Pennsylvania Trauma Outcome Study between 1993 and 1997 was conducted. Patients were stratified according to type of trauma center: PTC; Level I ATC; Level II ATC; or ATC with added qualifications (AQ). Functional outcome at discharge was analyzed. RESULTS: For severely injured children, there was an overall trend toward improved functional outcome at PTC compared with ATC AQ and ATC I, but no difference compared with ATC II. PTC showed improved functional outcome at discharge for head injury compared with ATC AQ and ATC I. CONCLUSION: There is an overall trend toward improved functional outcome at discharge for children treated at PTC compared with those treated at ATC AQ and ATC I. Improved outcome for head injury may be a key factor contributing to improved outcome at PTC.

Nitric oxide mediates dendritic cell apoptosis by downregulating inhibitors of apoptosis proteins and upregulating effector caspase activity.

BACKGROUND: Dendritic cells (DCs) play a crucial role in the amplification of the immune response by promoting antigen presentation, T-lymphocyte proliferation, and proinflammatory cytokine and nitric oxide (NO) production. We have previously shown that the exogenous NO donor, s-nitroso-N-acetyl-penicillamine, promotes DC apoptosis by disrupting the mitochondrial membrane potential, which induces cytochrome-C release and activates caspase 3. To further elucidate the signaling pathway, we examined the expression of cellular inhibitors of apoptosis proteins (cIAPs) and poly (ADP-ribose) polymerase cleavage (PARP), a terminal event in the apoptotic cascade. METHODS: DC2.4 were exposed to 250 micromol/L s-nitroso-N-acetyl-penicillamine for various intervals. Apoptosis and necrosis were measured by terminal deoxynucleotidyl transferase nick-end labeling assay or flow cytometry with Annexin V and propidium iodide. DC2.4 were cultured with the pan-caspase inhibitor, ZVAD (100 micromol/L). cIAP, pro-caspases, and PARP expression or activation was measured by Western blot. Caspase enzyme activity was confirmed with the use of specific substrates. RESULTS: NO-induced DC apoptosis correlated with the downregulation of cIAP expression. Caspase 3 and 6 were upregulated by SNAP and significantly inhibited by ZVAD. Maximal PARP cleavage occurred at 8 hours and coincided with the downregulation of cIAP and peak caspase 3 and near maximal caspase 6 activity. CONCLUSIONS: NO-induced DC apoptosis is associated with the downregulation of cIAP expression, which facilitates caspase cascade activation and subsequent PARP cleavage.

Intestinal cytokine gene expression in infants with acute necrotizing enterocolitis: interleukin-11 mRNA expression inversely correlates with extent of disease.

BACKGROUND/PURPOSE: The authors have shown previously that surgical specimens from infants with acute necrotizing enterocolitis (NEC) show upregulation of inducible nitric oxide (NO) synthase (iNOS) and interferon-gamma mRNA. However, the contribution of other inflammatory cytokines such as interleukin-8 (IL-8), IL-11, and IL-12 has not been defined. Likewise, the role of GTP-cyclohydrolase, the rate-limiting enzyme in tetrahydrobiopterin synthesis, and thus NO production by iNOS is unclear. In this study, the authors sought to further define the pattern of cytokine expression seen in infants with acute NEC. METHODS: The authors measured intestinal cytokine mRNA expression by semiquantitative reverse transcriptase polymerase chain reaction in 21 infants with histologically confirmed NEC, 18 with other inflammatory conditions, and in 9 patients without intestinal inflammation. Guanosine triphosphate-cyclohydrolase (GTP-CH) activity was measured by specific enzyme assay. Univariate exact logistic regression analysis was performed to identify predictors of outcome. RESULTS: IL-8 and IL-11 mRNA were upregulated in patients with acute NEC compared with those with other inflammatory conditions or those without disease; these levels returned to baseline at the time of stoma closure. Increased IL-11 mRNA decreased the likelihood of pan-necrosis (odds ratio, 0.93; P =.002). Increased IL-12 levels (but not IL-8) seemed to protect against pan-necrosis (odds ratio, 0.70; P =.06). CONCLUSIONS: Local upregulation of IL-11 may represent an adaptive response designed to limit the extent of intestinal damage in NEC. Decreased IL-12 levels may contribute to the pathogenesis of NEC by allowing bacteria to escape host defenses.

Driveway injuries in children: risk factors, morbidity, and mortality.

BACKGROUND/PURPOSE: Injuries that occur around the driveway are not typically regarded as reportable to the police and thus are often underrecognized. The aim of this study was to characterize the pattern and consequences of motor vehicle collisions that occur in the driveway. METHODS: Over the past 13 years, 64 patients admitted to the Children's Hospital of Pittsburgh sustained motor vehicle-related injuries in a driveway. These injuries resulted from a vehicle driven by an adult driver striking a child (group 1) or a child shifting an idle vehicle out of gear (group 2). We compared demographic variables and outcome measures between the 2 groups. RESULTS: There was no difference in gender, injury pattern, Injury Severity Score, length of stay, or operations performed between the groups. Patients in group 1 were younger, smaller, had a lower Glasgow Coma Scale, and had poorer outcomes. The majority of collisions (~65%) in group 1 resulted from a truck or sport-utility vehicle going in reverse. CONCLUSIONS: Younger children are more severely injured in driveway-related crashes, which are most likely to be caused by a truck or sport-utility vehicle going in reverse. These vehicles should be equipped with additional safety features such as extended mirrors to visualize small children.

Nitric oxide induces murine thymocyte apoptosis by oxidative injury and a p53-dependent mechanism.

Previously, we showed that NO induces thymocyte apoptosis via a caspase-1-dependent mechanism [(1) ]. In the present study, we investigated the role of heme oxygenase, catalase, bax, and p53 in this process. The NO donor, S-nitroso-N-acetyl penicillamine (SNAP), induced DNA fragmentation in thymocytes in a time- and concentration-dependent way. SNAP (100 microM) induced 50--60% apoptosis; higher doses did not increase the rate of apoptosis significantly. SNAP decreased catalase and heme iron (Fe) levels without affecting superoxide dismutase, glutathione, or total Fe stores in thymocytes. SNAP significantly increased the expression of heme oxygenase 1 (HSP-32), p53, and bax but not bcl-2. Treatment with the heme oxygenase inhibitor, tin protoporphyrin IX inhibited SNAP-induced thymocyte apoptosis. Furthermore, thymocytes from p53 null mice were resistant to NO-induced apoptosis. Our data suggest that NO may induce its cytotoxic effects on thymocytes by modulating heme oxygenase and catalase activity as well as up-regulating pro-apoptotic proteins p53 and bax.

Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge.

Sustained upregulation of inducible nitric oxide (NO) synthase in the liver after endotoxin [lipopolysaccharide (LPS)] challenge may result in hepatocellular injury. We hypothesized that administration of a NO scavenger, NOX, may attenuate LPS-induced hepatocellular injury. Sprague-Dawley rats received NOX or saline via subcutaneous osmotic pumps, followed 18 h later by LPS challenge. Hepatocellular injury was assessed using biochemical assays, light, and transmission electron microscopy (TEM). Interleukin (IL)-6 mRNA was measured by RT-PCR. Tumor necrosis factor (TNF)-alpha protein expression was determined by immunohistochemistry. NOX significantly reduced serum levels of ornithine carbamoyltransferase and aspartate aminotransferase. TNF-alpha and IL-6 expression were increased in the livers of saline-treated but not NOX-treated rats. Although there was no difference between groups by light microscopy, TEM revealed obliteration of the space of Disse in saline-treated but not in NOX-treated animals. Electron paramagnetic resonance showed the characteristic mononitrosyl complex in NOX-treated rats. We conclude that NOX reduces hepatocellular injury after endotoxemia. NOX may be useful in the management of hepatic dysfunction secondary to sepsis or other diseases associated with excessive NO production.

Development of a novel age-specific pediatric trauma score.

BACKGROUND/PURPOSE: Trauma scoring systems are needed to provide efficient triage of injured patients and to assess differences in outcomes and quality of care between different trauma centers. Current scoring systems used in pediatric trauma are not age specific, and thus have significant limitations. METHODS: The authors queried the Pennsylvania Trauma Outcome Study for all children 0 to 16 years entered in the database from 1993 to 1996. Age-specific threshold values for systolic blood pressure, pulse, and respiratory rate were established. Using coded scores for these age-specific values and Glasgow Coma Scale, an age-specific pediatric trauma score (ASPTS) was derived. Triage ASPTS (T-ASPTS) consisted of the integer sum of coded scores for the 4 variables, whereas ASPTS was calculated using weighted coefficients derived from logistic regression for each variable. RESULTS: T-ASPTS correlated with mortality rate. Using a threshold score of less than 10, T-ASPTS predicted mortality rate with a sensitivity of 96.97% and a specificity of 88.83%. T-ASPTS predicted mortality rate and percentage of patients with Injury Severity Score greater than 20 with similar sensitivity to the Revised Trauma Score (RTS), but T-ASPTS was more specific. The ASPTS predicted probability of survival more accurately than the RTS. CONCLUSIONS: ASPTS performs favorably as both a triage score and as a tool for predicting probability of survival for outcomes analysis. Further comparisons to existing trauma scores are needed to verify the utility of ASPTS.

Nitric oxide protects thymocytes from gamma-irradiation-induced apoptosis in correlation with inhibition of p53 upregulation and mitochondrial damage.

Apoptosis plays a crucial role in clonal deletion in the thymus, and NO has been shown to prevent apoptosis in some cell types. Therefore, we examined the effect of NO on gamma-irradiation-induced thymocyte apoptosis. Treatment of 5 Gy gamma-irradiated thymocytes with 1 mM SNAP reduced cell death from 78 to 49% after 8 h incubation (spontaneous cell death in medium control cells was 26%). Coincubation with ZVAD blocked both the spontaneous cell death and the cell death induced by SNAP or gamma-irradiation. The gamma-irradiation-induced increase in caspase 3 and 6 activities was inhibited in the presence of SNAP. The increase in cytosolic cytochrome c as well as the decrease in mitochondrial membrane potential after gamma-irradiation was inhibited in the presence of SNAP. SNAP treatment also decreased the p53 upregulation in gamma-irradiated cells. In summary, we found that NO exerts a protective effect on mouse thymocyte apoptosis induced by gamma-irradiation. The mechanism of this protective effect may involve inhibition of p53 upregulation and reduction in mitochondrial damage, with subsequent inhibition of downstream caspase activation.

Controversies in the management of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most frequent and lethal disease that affects the gastrointestinal tract of the premature infant. Controversy persists as to the most appropriate management once the diagnosis is confirmed. METHODS: Review of the pertinent medical literature. RESULTS: The incidence of NEC is increasing, but the survival rate is not. Initial management of NEC consists of bowel rest, orogastric decompression, intravenous hydration, and broad-spectrum antibiotics; surgical intervention is typically reserved for infants with advanced disease or evidence of intestinal perforation. There is no consensus in the literature regarding the optimal treatment strategy for patients who require surgical intervention. There exists a lack of randomized trials comparing definitive intestinal resection with or without primary anastomosis, intestinal diversion with limited resection, or peritoneal drainage without resection. CONCLUSION: An individualized approach must be taken to achieve optimum survival for patients with NEC. Isolated perforation, in our opinion, is best managed with resection and enterostomy, whereas pan-intestinal involvement is best managed with proximal diversion alone.

Risk factors and predictors of mortality in children after ejection from motor vehicle crashes.

PURPOSE: Mortality after ejection from a motor vehicle crash (MVC) has been studied extensively in adults. The magnitude of this problem in children is relatively unknown. We retrospectively examined fatalities resulting from ejection after MVC in the state of Pennsylvania to define risk factors and predictors of mortality in children. METHODS: The records for all patients 0 to 16 years of age involved in an MVC and entered in the Pennsylvania Trauma Outcome Study between 1993 and 1997 were reviewed. We examined mortality, length of hospitalization, major injuries sustained, and impact of safety restraint devices. Significant differences were determined using chi2 test. RESULTS: There were 2,298 children involved in MVCs during this period; 189 were ejected. A total of 77% of the ejected passengers were greater than 10 years of age, 16% were 0 to 4 years of age, and 7% were 5 to 9 years of age. Overall, 88% of the ejected occupants were unrestrained. Ejection nearly tripled the overall mortality rate and significantly increased the Injury Severity Score for each age group. Infants and children 0 to 4 years of age had the highest fatality rate despite having a lower Injury Severity Score than all other age groups. Head injuries accounted for the majority of deaths in all age groups. Children older than 10 years of age had a higher incidence of associated chest, abdominal, and pelvic injuries. CONCLUSION: Our data show that most children ejected from MVCs were either unrestrained or improperly restrained. Head injuries were the most common cause of death in all age groups. Greater public awareness through educational programs targeting parents and children at risk may reduce this serious problem.

Inhibition of NF-kappaB by IkappaB prevents cytokine-induced NO production and promotes enterocyte apoptosis in vitro.

Nuclear factor-kappaB (N-kappaB) plays a key role in gut inflammation. NF-kappaB up-regulates proinflammatory genes encoding cytokines, adhesion molecules, and inducible nitric oxide synthase (iNOS). However, NF-kappaB has also been shown to up-regulate protective or anti-apoptotic factors. We utilized an adenoviral vector carrying a super-repressor form of the inhibitor of NF-kappaB, IkappaB, to examine the effects of NF-kappaB inhibition on cytokine-induced nitric oxide production and apoptosis in rat small intestinal epithelial cells (IEC-6). Chemical inhibitors of NF-kappaB, including pyrrolidine dithiocarbamate (PDTC), tosyl-lysine-chloromethylketone (TLCK), genistein, and N-acetyl-leu-leu-norleucinal (n-LLnL) were also utilized. Treatment of AdIkappaB-transfected cells with cytomix [1000 U/mL IFN-gamma, 1 nM IL-1beta, and 10 ng/mL tumor necrosis factor alpha (TNFalpha)] or TNFalpha-containing cytokine combinations resulted in inhibition of cytokine-induced nitrite production and a marked increase in apoptosis compared to control cells. Apoptosis occurred independently of nitric oxide (NO) production since exogenous sources of NO did not inhibit apoptosis. Inducible NOS and clAP were down-regulated in AdIkappaB-transfected cells treated with cytomix. TLCK and LLnL treatment also induced apoptosis in cytomix-treated cells, while PDTC and genistein did not. Thus, although NF-kappaB up-regulates various pro-inflammatory genes, it may also have protective or anti-apoptotic effects in enterocytes. NF-kappaB appears necessary for upregulating cIAP in IEC-6 cells upon cytokine exposure.

Mechanisms of blunt perineal injury in female pediatric patients.

BACKGROUND/PURPOSE: Controversy persists regarding the causes of and the morbidity associated with blunt perineal injuries in children. The purpose of this study was to determine the most common mechanisms of blunt perineal trauma in female pediatric patients and to define the subset of patients that may benefit the most from an examination under anesthesia (EUA). METHODS: Nearly 4,450 female pediatric patients were entered in the Pennsylvania Trauma Outcome Study database between 1993 and 1997. The mechanism and extent of perineal injury, surgical repair, and associated injuries were examined for all girls 0 to 16 years of age with a diagnosis of blunt perineal trauma. RESULTS: A total of 358 girls experienced blunt perineal trauma. Motor vehicle crashes (MVC) accounted for the majority of injuries in all age groups. Falls and bicycle-related injuries were significantly more prevalent in children less than 9 years of age, and assaults in children 0 to 4 years. Head trauma was the most common associated injury in children less than 15 years. Children less than 10 years of age required surgical repair of their perineal injuries more frequently than their older counterparts. Perineal injuries caused by falls, assaults, or playground-related equipment were more likely to require surgical repair than those caused by other mechanisms. CONCLUSIONS: Perineal injuries that require surgical repair occur predominantly in patients less than 10 years of age who sustain blunt perineal trauma from a variety of causes, but rarely MVC. Thus, such patients should undergo aggressive evaluation, including EUA, especially if they present with perineal bleeding, hematoma, or swelling. Furthermore, perineal injuries in children under 4 years should raise the suspicion of abuse.

Impact of pediatric trauma centers on mortality in a statewide system.

BACKGROUND: Regional pediatric trauma centers (PTC) were established to optimize the care of injured children. However, because of the relative shortage of PTC, many injured children continue to be treated at adult trauma centers (ATC). As a result, a growing controversy has evolved regarding the impact of PTC and ATC on outcome for injured children. METHODS: A retrospective analysis of 13,351 injured children entered in the Pennsylvania Trauma Outcome Study between 1993 and 1997 was conducted. Patients were stratified according to mechanism of injury, injury severity, specific organ injury, and type of trauma center: PTC; Level I ATC (ATC I); Level II ATC (ATC II); or ATC with added qualifications to treat children (ATC AQ). Mortality was the major outcome variable measured. RESULTS: Most injured children were treated at a PTC or ATC AQ. The majority of children below 10 years of age were admitted to PTC. Patients treated at PTC and ATC had similar injury severity as determined by median Injury Severity Score, mean Revised Trauma Score, and Glasgow Coma Scale. Overall survival was significantly better at PTC and ATC AQ compared with ATC I and ATC II. Survival for head, spleen, and liver injuries was significantly better at PTC compared with ATC AQ, ATC I, or ATC II. Children who sustained moderate or severe head injuries were more likely to undergo neurosurgical intervention and have a better outcome when treated at a PTC. Despite similar mean Abbreviated Injury Scores for spleen and liver, significantly more children underwent surgical exploration (especially splenectomy) for spleen and liver injuries at ATC compared with PTC. CONCLUSION: Children treated at PTC or ATC AQ have significantly better outcome compared with those treated at ATC. Severely injured children (Injury Severity Score > 15) with head, spleen, or liver injuries had the best overall outcome when treated at PTC. This difference in outcome may be attributable to the approach to operative and nonoperative management of head, liver, and spleen injuries at PTC.

Recombinant human interleukin-11 prevents mucosal atrophy and bowel shortening in the defunctionalized intestine.

BACKGROUND: Mucosal atrophy and bowel shortening are the hallmark of proximal intestinal diversion for extensive necrotizing enterocolitis (NEC) or Thiry-Vella fistulas (TVF), in which the ends of a defunctionalized loop of intestine are exteriorized as stomas. Recombinant human interleukin-11 (rhIL-11) is a pleiotropic cytokine that promotes epithelial regeneration and enhances adaptation after bowel resection. The authors hypothesized that rhIL-11 may prevent mucosal atrophy and bowel shortening in rats with TVF METHODS: After creation of ileal TVF, Sprague-Dawley rats were selected randomly to receive either rhIL-11 or equal volume of 0.1% bovine serum albumin (BSA) subcutaneously daily. On day 14, the TVF were excised and examined morphologically. Enterocyte apoptosis was measured using the TUNEL assay. Mucosal DNA and protein content were measured. RESULTS: Administration of rhIL-11 resulted in a significantly greater weight gain and less shortening of TVF than BSA treatment. TVF from the rhIL-11-treated group showed evidence of hyperplasia and hypertrophy and increased crypt to villus ratio. The BSA group had substantial mucosal atrophy. There was a qualitative decrease in the incidence of apoptosis in the rhIL-11 group. CONCLUSIONS: Recombinant human IL-11 prevents mucosal atrophy and shortening of defunctionalized intestinal loops. It may help reduce the incidence of short gut syndrome in infants with extensive NEC.

Nitric oxide induces thymocyte apoptosis via a caspase-1-dependent mechanism.

We previously showed that NO induces apoptosis in thymocytes via a p53-dependent pathway. In the present study, we investigated the role of caspases in this process. The pan-caspase inhibitor, ZVAD-fmk, and the caspase-1 inhibitor, Ac-YVAD-cho, both inhibited NO-induced thymocyte apoptosis in a dose-dependent manner, whereas the caspase-3 inhibitor, Ac-DEVD-cho, had little effect even at concentrations up to 500 microM. ZVAD-fmk and Ac-YVAD-cho were able to inhibit apoptosis when added up to 12 h, but not 16 h, after treatment with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). Caspase-1 activity was up-regulated at 4 h and 8 h and returned to baseline by 24 h; caspase-3 activity was not detected. Cytosolic fractions from SNAP-treated thymocytes cleaved the inhibitor of caspase-activated deoxyribonuclease. Such cleavage was completely blocked by Ac-YVAD-cho, but not by Ac-DEVD-cho or DEVD-fmk. Poly(ADP-ribose) polymerase (PARP) was also cleaved in thymocytes 8 h and 12 h after SNAP treatment; addition of Ac-YVAD-cho to the cultures blocked PARP cleavage. Furthermore, SNAP induced apoptosis in 44% of thymocytes from wild-type mice; thymocytes from caspase-1 knockout mice were more resistant to NO-induced apoptosis. These data suggest that NO induces apoptosis in thymocytes via a caspase-1-dependent but not caspase-3-dependent pathway. Caspase-1 alone can cleave inhibitor of caspase-activated deoxyribonuclease and lead to DNA fragmentation, thus providing a novel pathway for NO-induced thymocyte apoptosis.

Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.

BACKGROUND: Previous investigators have relied on administration of pro-inflammatory cytokines or invasive surgical procedures to reproduce the morphologic changes of necrotizing enterocolitis (NEC) in rats. However, these artificial insults do not mimic the human disease. We developed a reproducible model of NEC in rats that more closely resembles human NEC and determined the pattern of inflammatory cytokine expression in this model. MATERIALS AND METHODS: Newborn rats were randomized into four groups. Groups 1 and 2 were breast-fed, while Groups 3 and 4 were gavaged with formula thrice daily. In addition, Groups 2 and 4 were subjected to 3 min of hypoxia thrice daily, prior to each feeding. The rats were killed on day 4 and the distal 2 cm of terminal ileum was harvested for morphological studies and analysis of inflammatory cytokine mRNA expression. RESULTS: Nearly 70% of formula-fed neonatal rats displayed moderate or severe morphological abnormalities resembling human NEC. Breast-fed pups had normal histology. The terminal ileum from rats with abnormal histology demonstrated increased inducible nitric oxide synthase (iNOS) expression, decreased interleukin-12 (IL-12) mRNA expression, and enterocyte apoptosis. There was a trend toward upregulation of IFN-gamma mRNA, but no difference in expression of TNF-alpha mRNA. Hypoxia did not significantly alter intestinal morphology or mRNA expression. CONCLUSIONS: Formula-fed neonatal rats, with or without hypoxia, exhibit morphological changes in the intestinal epithelium similar to those seen in patients with acute NEC. The mechanism likely involves upregulation of iNOS mRNA, enterocyte apoptosis, and decreased IL-12 production in the intestinal epithelium. This model may offer a simple reproducible method for inducing experimental NEC.