RESUMO
BackgroundThe mechanisms underlying aberrant activation of intestinal Toll-like receptor 4 (TLR4) signaling in necrotizing enterocolitis (NEC) remain unclear. In this study, we examined the role of single-immunoglobulin interleukin-1 receptor-related molecule (SIGIRR), an inhibitor of TLR signaling, in modulating experimental NEC vulnerability in mice.MethodsExperimental NEC was induced in neonatal wild-type and SIGIRR-/- mice using hypoxia, formula-feeding, and lipopolysaccharide administration. Intestinal TLR canonical signaling, inflammation, apoptosis, and severity of experimental NEC were examined at baseline and after NEC induction in mice.ResultsSIGIRR is developmentally regulated in the neonatal intestine with a restricted expression after birth and a gradual increase by day 8. At baseline, breast-fed SIGIRR-/- mouse pups exhibited low-grade inflammation and TLR pathway activation compared with SIGIRR+/+ pups. With experimental NEC, SIGIRR-/- mice had significantly more intestinal interleukin (IL)-1ß, KC (mouse homolog to IL-8), intercellular adhesion molecule-1 (ICAM-1), and interferon-beta (IFN-ß) expression in association with the amplified TLR pathway activation. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, cleaved caspase 3, and severity of intestinal injury with NEC were worse in SIGIRR-/- mice in comparison with SIGIRR+/+ mice.ConclusionSIGIRR is a negative regulator of TLR4 signaling in the developing intestine, and its insufficiency results in native intestinal TLR hyper-responsiveness conducive to the development of severe experimental NEC in mice.
Assuntos
Enterocolite Necrosante/metabolismo , Receptores de Interleucina-1/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Citocinas/metabolismo , Modelos Animais de Doenças , Hipóxia , Imunidade Inata , Inflamação , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Fosforilação , Transdução de SinaisRESUMO
BACKGROUND: Intestinal alkaline phosphatase (IAP) activity is decreased in necrotizing enterocolitis (NEC), and IAP supplementation prevents NEC development. It is not known if IAP given after NEC onset can reverse the course of the disease. We hypothesized that enteral IAP given after NEC induction would not reverse intestinal injury. MATERIALS AND METHODS: NEC was induced in Sprague-Dawley pups by delivery preterm followed by formula feedings with lipopolysaccharide (LPS) and hypoxia exposure and continued up to 4 d. IAP was added to feeds on day 2 until being sacrificed on day 4. NEC severity was scored based on hematoxylin and eosin-stained terminal ileum sections, and AP activity was measured using a colorimetric assay. IAP and interleukin-6 expression were measured using real time polymerase chain reaction. RESULTS: NEC pups' alkaline phosphatase (AP) activity was decreased to 0.18 U/mg compared with controls of 0.57 U/mg (P < 0.01). Discontinuation of LPS and hypoxia after 2 d increased AP activity to 0.36 U/mg (P < 0.01). IAP supplementation in matched groups did not impact total AP activity or expression. Discontinuing LPS and hypoxia after NEC onset improved intestinal injury scores to 1.14 compared with continued stressors, score 2.25 (P < 0.01). IAP supplementation decreased interleukin-6 expression two-fold (P < 0.05), though did not reverse NEC intestinal damage (P = 0.5). CONCLUSIONS: This is the first work to demonstrate that removing the source of NEC improves intestinal damage and increases AP activity. When used as a rescue treatment, IAP decreased intestinal inflammation though did not impact injury making it likely that IAP is best used preventatively to those neonates at risk.
Assuntos
Fosfatase Alcalina/uso terapêutico , Enterocolite Necrosante/tratamento farmacológico , Intestinos/enzimologia , Fosfatase Alcalina/metabolismo , Animais , Animais Recém-Nascidos , Avaliação Pré-Clínica de Medicamentos , Enterocolite Necrosante/patologia , Feminino , Interleucina-6/metabolismo , Intestinos/patologia , Reação em Cadeia da Polimerase , Gravidez , Ratos Sprague-DawleyRESUMO
Hyphema is rarely seen in neonates. Although most cases are secondary to instrument-assisted delivery, neonatal hyphema can occur spontaneously or result from an underlying coagulopathy. We report the case of an infant who was born with unilateral hyphema and was subsequently found to have gestational alloimmune liver disease-a condition where maternal antibodies attack the infant's liver, leading to a hypocoagulable state. Our patient was treated with topical prednisolone and cyclopentolate/phenylephrine, with subsequent resolution of the hyphema.
Assuntos
Glucocorticoides , Hifema , Humanos , Hifema/etiologia , Hifema/diagnóstico , Hifema/tratamento farmacológico , Recém-Nascido , Feminino , Glucocorticoides/uso terapêutico , Gravidez , Prednisolona/uso terapêutico , Ciclopentolato/uso terapêutico , Ciclopentolato/administração & dosagem , Hepatopatias/diagnóstico , Hepatopatias/etiologia , Hepatopatias/imunologia , Midriáticos/uso terapêutico , Midriáticos/administração & dosagem , Masculino , Quimioterapia CombinadaRESUMO
BACKGROUND: Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with an incidence of 0.5-2.4 cases per 1000 live births and a mortality rate between 10% and 50%. Neonates affected by NEC develop a septic injury that is associated with increased risk of neurological impairment due to intraventricular bleeding and chronic lung disease. Intestinal alkaline phosphatase (IAP) is an endogenous protein that has been shown to inactivate the endotoxin lipopolysaccharide (LPS), and has recently been used successfully as an adjunct to treat sepsis in adult patients. We tested the hypothesis that systemic, exogenous IAP will mitigate the inflammatory response as measured by serum levels of proinflammatory cytokines in a rat model of NEC. METHODS: Newborn Sprague-Dawley rats were divided into groups. Control pups were dam fed. NEC was induced by feeding formula containing LPS and exposure to intermittent hypoxia. NEC pups were given intraperitoneal injections of 4 or 40 glycine units (U) of IAP or placebo twice daily. Intestine and serum was collected for cytokine analysis as well as measurement of alkaline phosphatase activity. RESULTS: Systemic IAP administration significantly increased serum alkaline phosphatase activity in a dose- and time-dependent fashion. The proinflammatory cytokines tumor necrosis factor α, interleukin 6, and interleukin 1ß were significantly increased in NEC rats versus controls on days 2 and 3. Importantly, treatment with 40 U systemic IAP decreased these proinflammatory cytokines back to near-control levels. CONCLUSIONS: Systemic IAP administration appears effective in mitigating the systemic inflammatory response associated with NEC, and may prove to be a valuable adjunctive treatment for NEC.
Assuntos
Fosfatase Alcalina/uso terapêutico , Enterocolite Necrosante/tratamento farmacológico , Intestinos/enzimologia , Síndrome de Resposta Inflamatória Sistêmica/prevenção & controle , Fosfatase Alcalina/sangue , Animais , Animais Recém-Nascidos , Citocinas/sangue , Enterocolite Necrosante/imunologia , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Supplementation of intestinal alkaline phosphatase (IAP), an endogenous protein expressed in the intestines, decreases the severity of necrotizing enterocolitis (NEC)-associated intestinal injury and permeability. We hypothesized that IAP administration is protective in a dose-dependent manner of the inflammatory response in a neonatal rat model. MATERIALS AND METHODS: Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed on day of life 3. Control pups were vaginally delivered and dam fed. Preterm pups were delivered via cesarean section and exposed to intermittent hypoxia and formula feeds containing lipopolysaccharide (NEC) with and without IAP. Three different standardized doses were administered to a group of pups treated with 40, 4, and 0.4U/kg of bovine IAP (NEC+IAP40, IAP4, or IAP0.4U). Reverse transcription-real-time polymerase chain reaction (RT-PCR) for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α on liver and lung tissues and serum cytokine analysis for interleukin (IL)-1ß, IL-6, IL-10, and TNF-α were performed. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests, expressed as mean±standard error of the mean and P≤0.05 considered significant. RESULTS: Levels of cytokines IL-1ß, IL-6, and TNF-α increased significantly in NEC versus control, returning to control levels with increasing doses of supplemental enteral IAP. Hepatic and pulmonary TNF-α and iNOS messenger ribonucleic acid expressions increased in NEC, and the remaining elevated despite IAP supplementation. CONCLUSIONS: Proinflammatory cytokine expression is increased systemically with intestinal NEC injury. Administration of IAP significantly reduces systemic proinflammatory cytokine expression in a dose-dependent manner. Early supplemental enteral IAP may reduce NEC-related injury and be useful for reducing effects caused by a proinflammatory cascade.
Assuntos
Fosfatase Alcalina/uso terapêutico , Citocinas/sangue , Enterocolite Necrosante/prevenção & controle , Animais , Animais Recém-Nascidos , Avaliação Pré-Clínica de Medicamentos , Enterocolite Necrosante/sangue , Enterocolite Necrosante/complicações , Feminino , Hepatite/etiologia , Hepatite/prevenção & controle , Pneumonia/etiologia , Pneumonia/prevenção & controle , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with a mortality rate between 10 and 50%. The onset of necrotizing enterocolitis is highly variable and associated with numerous risk factors. Prior research has shown that enteral supplementation with intestinal alkaline phosphatase (IAP) decreases the severity of NEC. The aim of this study is to investigate whether IAP is protective to the preterm intestine in the presence of formula feeding and in the absence of NEC. METHODS: Preterm rat pups were fed formula with or without supplementation with IAP, and intestine was obtained on day of life 3 for analysis of IAP activity, mRNA expression of TNFα, IL-6 and iNOS and permeability and cytokine expression after LPS exposure. RESULTS: There was no difference in the absolute and intestine specific alkaline phosphatase activity in both groups. Rat pups fed IAP had decreased mRNA expression of the inflammatory cytokines TNFα, IL-6 and iNOS. Pups supplemented with IAP had decreased permeability and inflammatory cytokine expression after exposure to LPS ex vivo when compared to formula fed controls. CONCLUSIONS: Our results support that IAP is beneficial to preterm intestine and decreases intestinal injury and inflammation caused by LPS.
Assuntos
Fosfatase Alcalina/administração & dosagem , Enterocolite Necrosante/tratamento farmacológico , Mucosa Intestinal/metabolismo , Administração Oral , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Animais Recém-Nascidos , Citocinas/biossíntese , Citocinas/genética , Modelos Animais de Doenças , Enterocolite Necrosante/genética , Enterocolite Necrosante/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Necrotizing enterocolitis (NEC) is a complication of prematurity. The etiology is unknown, but is related to enteral feeding, ischemia, infection, and inflammation. Reactive oxygen species production, most notably superoxide, increases in NEC. NADPH oxidase (NOX) generates superoxide, but its activity in NEC remains unknown. We hypothesize that NOX-derived superoxide production increases in NEC. Newborn Sprague-Dawley rats were divided into control, formula-fed, formula/LPS, formula/hypoxia, and NEC (formula, hypoxia, and LPS). Intestinal homogenates were analyzed for NADPH-dependent superoxide production. Changes in superoxide levels on days 0-4 were measured. Inhibitors for nitric oxide synthase (L-NAME) and NOX2 (GP91-ds-tat) were utilized. RT-PCR for eNOS, NOX1, GP91phox expression was performed. Immunofluorescence studies estimated the co-localization of p47phox and GP91phox in control and NEC animals on D1, D2, and D4. NEC pups generated more superoxide than controls on D4, while all other groups were unchanged. NADPH-dependent superoxide production was greater in NEC on days 0, 3, and 4. GP91-ds-tat decreased superoxide production in both groups, with greater inhibition in NEC. L-NAME did not alter superoxide production. Temporally, superoxide production varied minimally in controls. In NEC, superoxide generation was decreased on day 1, but increased on days 3-4. GP91phox expression was higher in NEC on days 2 and 4. NOX1 and eNOS expression were unchanged from controls. GP91phox and p47phox had minimal co-localization in all control samples and NEC samples on D1 and D2, but had increased co-localization on D4. In conclusion, this study proves that experimentally-induced NEC increases small intestinal NOX activity. All components of NEC model are necessary for increased NOX activity. NOX2 is the major source, especially as the disease progresses.
Assuntos
Modelos Animais de Doenças , Enterocolite Necrosante/enzimologia , Intestinos/enzimologia , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Animais , Animais Recém-Nascidos , Nutrição Enteral , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/patologia , Inibidores Enzimáticos/farmacologia , Feminino , Imunofluorescência , Hipóxia/complicações , Hipóxia/fisiopatologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Glicoproteínas de Membrana/genética , NADPH Oxidase 2 , NADPH Oxidases/genética , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
INTRODUCTION: Inflammation in the premature intestine is a key factor that leads to the development of necrotizing enterocolitis (NEC). Activation of nuclear factor kappa B (NF-κB) and subsequent inflammation increases the severity of NEC. The aim of this study was to investigate the early temporal expression of inflammatory markers and activation of NF-κB in a neonatal rat model of NEC. METHODS: Pre- and full-term newborn Sprague-Dawley rat pups were sacrificed at birth, 1.5, 4, 8, and 24 hours after receiving their first feed. Control pups were vaginally delivered and mother fed; NEC was induced by a combination of gavage feeding formula, hypoxia, and enteral lipopolysaccharide (LPS); and formula fed pups were fed every 4 hours with infant formula. Ileal tissue was collected for immunohistochemistry, real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Serum was collected for cytokine content. Fold change of expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α), IL-10, NF-κB p65, and IκBα used RT-PCR. Data were analyzed by paired two-tailed t test, expressed as mean ± standard error of the mean, and p ≤ 0.05 considered significant. RESULTS: No histologic injury was evident in ileal sections. At 1.5 h, iNOS expression increased twofold over control in NEC pups (2.1 vs. 1.0, p ≤ 0.05) and remained elevated at 24 h (0.7 vs. 9.4, p ≤ 0.05). IL-1ß and IL-6 reached a peak at 24 h in NEC tissue compared with control. IL-10 expression rose in NEC pups after 4 h of insult and remained elevated in formula and NEC stressed pups. Coincident with an increase in p65 translocation into the nucleus and a reduction of IκBα detected in the cytoplasm, increased transcription of IκBα occurs. CONCLUSION: These findings suggest that NF-κB activation initiates inflammation early in the course of NEC resulting in increased proinflammatory protein expression, underscoring the importance of the inflammatory response in this NEC model, which precedes evidence of histological injury.
Assuntos
Citocinas/sangue , Nutrição Enteral/efeitos adversos , Enterocolite Necrosante/etiologia , Íleo/metabolismo , Fórmulas Infantis , Estresse Fisiológico/fisiologia , Fator de Transcrição RelA/metabolismo , Animais , Biomarcadores/metabolismo , Enterocolite Necrosante/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Hipóxia , Imuno-Histoquímica , Recém-Nascido , Lipopolissacarídeos , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TempoRESUMO
BACKGROUND: Previously, we have shown that supplementation of intestinal alkaline phosphatase (IAP) decreased severity of necrotizing enterocolitis (NEC)-associated intestinal injury. We hypothesized that IAP administration is protective of intestinal epithelial barrier function in a dose-dependent manner. METHODS: Control rat pups were vaginally delivered and breast-fed. Premature rats were divided into 4 groups: formula fed with lipopolysaccharide and hypoxia (NEC) or additional daily bovine IAP 40, 4, or 0.4 U/kg (NEC + IAP 40 U, IAP 4 U, or IAP 0.4 U). RESULTS: Necrotizing enterocolitis is associated with decreased IAP protein expression and activity. Supplemental IAP increases IAP activity in intestinal homogenates and decreased NEC injury score in a dose-dependent manner. Intestinal injury as measured by fluorescein isothiocyanate-dextran flux from ileal loops showed increased permeability vs control, but supplemental IAP reversed this. Tight junction proteins claudin-1, claudin-3, occludin, and zonula occludin 1 were elevated in the NEC and IAP-treated groups with differences in expression patterns. No differences in messenger RNA levels were observed on postinjury day 3. Intestinal alkaline phosphatase administration decreases intestinal NEC injury in a dose-dependent manner. CONCLUSION: Early enteral supplemental IAP may reduce NEC-related injury and may be useful for preserving the intestinal epithelial barrier function.