Probiotic and prebiotic supplementation for the prevention of neonatal necrotizing enterocolitis.

The pathophysiology of necrotizing enterocolitis (NEC) has not been clearly elucidated, but recent studies support the role of unbalanced pro-inflammatory signaling, leading to intestinal necrosis in premature infants. Although breast milk feeding is thought to reduce the risk of this condition, there are no preventive or therapeutic approaches that have consistently shown to be effective for this common and devastating disease. Recent studies show that probiotic colonization is abnormal in preterm neonates, and enteral supplementation with a variety of probiotic organisms can reduce the risk of disease. This chapter summarizes the current state-of-the-art regarding probiotics and NEC, but suggests caution until appropriately regulated products are available for use in this high-risk population.

Protective effect of bilirubin in ischemia-reperfusion injury in the rat intestine.

BACKGROUND: Although bilirubin, which crosses the blood-brain barrier, can cause irreversible brain damage, it also possesses antioxidant properties that may be protective against oxidative stress. Intestinal ischemia-reperfusion (IR) injury results in cell destruction, mediated via the generation of reactive oxygen species. Although increased serum bilirubin is correlated with increased antioxidant potential in the face of hyperoxia, evidence of bilirubin-associated protective effect against IR injury remains nonspecific. We therefore sought to investigate whether hyperbilirubinemia would be protective against IR injury to the intestine. METHODS: Young adult rats were randomly assigned to one of three groups: 1) IR/control (n = 12); 2) IR/hyperbilirubinemia (n = 10), in which IR was generated while the rats were treated with a continuous infusion of bilirubin; and 3) hyperbilirubinemia controls (n = 10). Blood and intestinal tissue samples were obtained to determine serial thiobarbituric acid reducing substances (index of lipid peroxidation) and for xanthine oxidase/xanthine dehydrogenase and glutathione/glutathione disulfide ratios. Intestinal histopathology was graded from 1 (normal) to 4 (severe necrotic lesions). RESULTS: Histopathologic scoring and circulating and tissue thiobarbituric acid reducing substances were highest in the IR/control animals compared with either the IR/hyperbilirubinemics or the controls. All of these are consistent with the most severe injury in this group. Xanthine oxidase/xanthine dehydrogenase ratios were not significantly different among the groups. CONCLUSION: Hyperbilirubinemia ameliorates the extent of intestinal IR injury in our model and appears to act as an antioxidant. This study supports the concept that bilirubin possesses some beneficial properties in vivo, although no direct clinical conclusions can be drawn from these data.

The role of polyunsaturated fatty acid supplementation in intestinal inflammation and neonatal necrotizing enterocolitis.

Dietary polyunsaturated fatty acid (PUFA) supplementation has been shown to reduce the incidence of necrotizing enterocolitis (NEC) in a recent randomized, controlled trial. These compounds are known to modulate the inflammatory cascade and to influence intestinal health in a variety of ways. Although the pathophysiology of NEC is not well understood, recent evidence suggests that platelet-activating factor (PAF) is a key endogenous mediator of intestinal necrosis in animals. Using a neonatal rat model of NEC that includes the key risk factors of asphyxia and formula feeding, we investigated the role of dietary PUFA supplementation on the incidence and pathophysiology of NEC. Our findings suggest that PUFA reduce the incidence of NEC by modulating PAF metabolism and endotoxin translocation.

Effect of polyunsaturated fatty acid (PUFA) supplementation on intestinal inflammation and necrotizing enterocolitis (NEC) in a neonatal rat model.

Inasmuch as long-chain polyunsaturated fatty acids (PUFA, metabolites of the essential n-3 and n-6 fatty acids) are known to modulate inflammation, we hypothesized that supplementation of formula with these compounds would prevent necrotizing enterocolitis (NEC) and intestinal inflammation in our neonatal rat model. Newborn rats were stressed with asphyxia and formula feeding, and randomly assigned to control formula, control with PUFA supplementation, and PUFA with nucleotides. Animals were followed for 72--96 h and assessed for death, gross and histologic NEC, intestinal apoptosis, endotoxemia, and intestinal mRNA synthesis of phospholipase A(2)-II (rate-limiting enzyme for platelet activating factor production), platelet activating factor receptor, and inducible nitric oxide synthase. We found that PUFA reduced the incidence of death and NEC compared with the other groups (NEC 8 of 24 versus 17 of 24 control and 13 of 23 PUFA + nucleotides, p < 0.05). Furthermore, PUFA reduced plasma endotoxemia at 48 h (25 +/- 4 EU/mL versus 276 +/- 39 EU/mL in control and 170 +/- 28 EU/mL in PUFA + nucleotide), intestinal phospholipase A(2)-II expression at 24 h, and platelet activating factor receptor expression at 48 h. Formula supplementation had no effect on apoptosis of intestinal epithelium or intestinal inducible nitric oxide synthase expression. Addition of nucleotides with PUFA abrogated the beneficial effects of PUFA on intestinal inflammation. We conclude that PUFA reduces the incidence of NEC and intestinal inflammation in a neonatal rat model.

New concepts in necrotizing enterocolitis.

Necrotizing enterocolitis is an overwhelming gastrointestinal emergency that primarily afflicts premature infants born weighing less than 1500 g. Despite years of investigation, the etiology remains unclear, and accepted prevention and treatment strategies are lacking. Studies published over the last year have provided new insight into several aspects of this complex disease. In this review, novel information is presented on (1) the epidemiology; (2) methods of early diagnosis, such as abdominal magnetic resonance imaging; (3) the importance of risk factors, including assessment of feeding strategies and role of bacterial colonization; (4) the pathophysiology, highlighting experimental and clinical trials evaluating the role of inflammatory mediators and growth factors on the disease; (5) preventive strategies, such as anaerobic bacterial supplementation; and (6) surgical interventions, including peritoneal drainage. Understanding some of these important aspects of necrotizing enterocolitis may help improve the outlook of patients with this dreaded disease. Although the incidence of neonatal necrotizing enterocolitis (NEC) and the mortality stemming from this disease have not significantly improved over the last 30 years, there is exciting new information that may significantly improve the outlook of patients with this overwhelming intestinal emergency in the near future.

Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model.

BACKGROUND & AIMS: Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of premature infants partly caused by intestinal bacterial proliferation. Because bifidobacteria are thought to reduce the risk for intestinal disturbances associated with pathogenic bacterial colonization, we hypothesized that exogenous bifidobacterial supplementation to newborn rats would result in intestinal colonization and a reduction in the incidence of neonatal NEC. METHODS: Newborn rat pups were given Bifidobacterium infantis (10(9) organisms per animal daily), Escherichia coli, or saline control and exposed to the NEC protocol consisting of formula feeding (Esbilac; 200 cal. kg(-1). day(-1)) and asphyxia (100% N(2) for 50 seconds followed by cold exposure for 10 minutes). Outcome measures included stool and intestinal microbiological evaluation, gross and histological evidence of NEC, plasma endotoxin concentration, intestinal phospholipase A(2) expression, and estimation of intestinal mucosal permeability. RESULTS: Bifidobacterial supplementation resulted in intestinal colonization by 24 hours and appearance in stool samples by 48 hours. Bifidobacteria-supplemented animals had a significant reduction in the incidence of NEC compared with controls and E. coli-treated animals (NEC, 7/24 B. infantis vs. 19/27 control vs. 16/23 E. coli; P < 0.01). Plasma endotoxin and intestinal phospholipase A(2) expression were lower in bifidobacteria-treated pups than in controls, supporting the role of bacterial translocation and activation of the inflammatory cascade in the pathophysiology of NEC. CONCLUSIONS: Intestinal bifidobacterial colonization reduces the risk of NEC in newborn rats.

Amelioration of ischemia-reperfusion injury in rat intestine by pentoxifylline-mediated inhibition of xanthine oxidase.

BACKGROUND: Intestinal ischemia-reperfusion (IR) injury results in cell destruction, which may be mediated by the generation of reactive oxygen species, potentially toxic metabolites of xanthine oxidase. Pentoxifylline (PTX) possesses a variety of biochemical and antioxidant properties that can improve capillary flow and tissue oxygenation. Because of these combined effects, it has been hypothesized that pentoxifylline would protect against intestinal IR. METHODS: Young adult rats were randomly assigned to one of four experimental groups: IR/Placebo (n = 12) in which superior and inferior mesenteric arteries were clamped for 45 minutes and then reopened; IR/PTX (n = 11) in which IR was induced as in the Placebo group, but with 25 mg/kg PTX at 0, 30, and 60 minutes; No IR/Placebo (n = 12); and No IR/PTX (n = 6) in which placebo and PTX were applied with no IR. Blood and intestinal samples were taken for serial thiobarbituric acid-reducing substances (TBARS; index of lipid peroxidation), for xanthine oxidase-xanthine dehydrogenase ratios, glutathione, myeloperoxidase, and histopathology. RESULTS: Animals in the IR/PTX group had lower TBARS and the least severe histopathologic injury. Xanthine oxidasexanthine dehydrogenase ratios were elevated only in IR/ Placebo (0.67+/-0.22 vs. 0.45+/-0.14 in IR/PTX; 0.42+/-0.22 in No IR/Placebo; and 0.40+/-0.11 in No IR/PTX; p = 0.0009). Reduced glutathione was diminished in IR/PTX animals (38.9 +/-1.35 vs. 46.1+/-7.0 in IR/Placebo; 41.1+/-2.5 in No IR/ Placebo; 43.6+/-1.0 in No IR/PTX; p = 0.048). No differences were recorded in myeloperoxidase levels among groups. CONCLUSIONS: Pentoxifylline ameliorates histopathologic signs of injury and decreases lipid peroxidation (TBARS). Normal xanthine oxidase-xanthine dehydrogenase ratios in the treated compared with IR-only animals imply that the protective effect of PTX is at least partially mediated through inhibition of xanthine oxidase.

The role of recombinant platelet-activating factor acetylhydrolase in a neonatal rat model of necrotizing enterocolitis.

Previous studies have shown that the endogenous inflammatory mediator platelet-activating factor (PAF) plays an important role in the pathophysiology of neonatal necrotizing enterocolitis (NEC). This study was designed to investigate the role of the PAF-degrading enzyme acetylhydrolase (PAF-AH) in a neonatal rat model of NEC. To study the absorption, localization, and activity of human recombinant PAF-AH (rPAF-AH), newborn rats were treated with enteral rPAF-AH, and plasma and intestines were sampled at 8 and 24 h for determination of PAF-AH enzyme activity and rPAF-AH concentration using a specific enzyme-linked immunoassay. To study the effect of rPAF-AH on neonatal NEC, rats were treated with rPAF-AH via the enteral route every 3 h, and then subjected to formula feeding and asphyxia per an established neonatal rat protocol for NEC. Pretreatment with enteral rPAF-AH significantly reduced the incidence of NEC compared with controls (6/26 versus 19/26, p < 0.001). We found that enteral rPAF-AH administration resulted in significant intestinal PAF-AH activity but no circulating PAF-AH activity despite immunohistochemical localization of the administered rPAF-AH to the intestinal epithelial cells. These findings suggest that rPAF-AH is functional and stable in the gut of neonatal rats. We conclude that enteral administration of rPAF-AH remains locally active and reduces the incidence of NEC in our experimental animal model.

Endothelin-1-induced placental and fetal growth restriction in the rat.

The objective of this study was to evaluate the effect of increased circulating endothelin on fetal and placental growth in the rat. Indwelling arterial and venous catheters were placed on day 14 of a 22-day gestation in timed-pregnant Sprague-Dawley rats. Saline, 0.2 nmol/kg/h endothelin, or 0.5 nmol/kg/h endothelin was continuously infused from day 15 through 21 in randomly assigned animals (n = 12 in each group). Maternal arterial blood pressure and serum endothelin levels were evaluated on days 15, 18, and 21 of gestation. On day 21, pregnancy outcome data including fetal and placental weights, litter size, and the occurrence of stillbirths were ascertained, and fetal blood was obtained for serum endothelin levels. All data were compared among the three groups, and statistical significance was determined by analysis of variance. Endothelin infusion resulted in a dose-dependent decrease in fetal and placental weights when compared to saline-treated pregnancies. A significant increase in maternal arterial blood pressure was noted only in the 0.5 nmol/kg/h endothelin group. Fetal and placental growth restriction occurred in the absence of maternal hypertension in the 0.2 nmol/kg/h group. These results demonstrate that endothelin infusion causes restriction of fetal and placental growth, even in the absence of maternal hypertension.

The platelet-activating factor receptor antagonist WEB 2170 prevents neonatal necrotizing enterocolitis in rats.

UNLABELLED: To evaluate the role of platelet-activating factor (PAF) in a neonatal rat model of necrotizing enterocolitis (NEC). METHODS: NEC was reproduced in newborn rats following exposure to formula feeding, asphyxia, and bacterial colonization. The role of endogenous PAF in neonatal NEC was studied by pretreating animals with PAF receptor antagonists (WEB 2170 and WEB 2086) and by measuring intestinal PAF content in animals with NEC, WEB-treated animals, and controls. RESULTS: We found that WEB 2170 (dosed using 10 mg/kg in a.m. and 30 mg/kg in p.m.) markedly reduced the incidence of NEC (3/17 vs. 14/18 control: p < 0.001) and death (6/17 vs. 17/18 control; p < 0.001) compared with saline-treated animals. Although lower WEB 2170 doses prevented NEC, neither fourfold higher dosing with WEB 2170 nor similar dosing with WEB 2086 affected the incidence of disease in this study. PAF content in intestine was elevated in NEC animals (270 +/- 80 pg/g) compared with WEB 2170-treated animals (0 pg/g) and maternally fed controls (70 +/- 50 pg/g). CONCLUSIONS: The data support the role of PAF in the final common pathway of neonatal NEC.

Endothelin receptor A antagonism prevents hypoxia-induced intrauterine growth restriction in the rat.

OBJECTIVE: Our purpose was to investigate the hypothesis that endothelin plays a critical role in maternal hypoxia-induced intrauterine growth restriction. STUDY DESIGN: Chronic indwelling venous and arterial catheters were placed on day 17 of a 22-day gestation in timed-pregnant Sprague-Dawley rats. Twelve rats were infused with saline solution and 12 with 6 mg/kg per day FR139317, an endothelin receptor A-specific antagonist. For gestational days 18 to 21 half the rats in each infusion group were housed in a normoxic environment and the other half in a hypoxic (14% oxygen) environment. On day 21 an arterial blood gas level was determined, the rats were then anesthetized, and a hysterotomy was performed. The weight of each pup and its corresponding placenta was recorded. Statistical significance was determined by analysis of variance. RESULTS: Among the rats receiving saline solution infusions, fetal weights were 20% less and placental weights were 11% less for those housed in a hypoxic environment compared with those housed in a normoxic environment (p < 0.003). Among the rats receiving FR139317 infusions, fetal and placental weights were not significantly different for those in a hypoxic environment compared with those in a normoxic environment. The fetal and placental weights for the rats receiving FR139317 infusion in hypoxic or normoxic environments were similar to those receiving saline solution in a normoxic environment. CONCLUSIONS: Endothelin plays a critical role in hypoxia-induced intrauterine growth restriction. Infusion of an endothelin antagonist prevents the intrauterine growth restriction caused by chronic hypoxia.

Evaluation of nitric oxide as a mediator of severe preeclampsia.

OBJECTIVE: Our purpose was to determine whether a reduction in nitric oxide synthesis occurs in women with severe preeclampsia as a consequence of soluble serum factors. STUDY DESIGN: Circulating nitrate and nitrite levels were compared between women who met standard clinical criteria for severe preeclampsia (n = 21) and maternal or gestational age-matched, normotensive, primagravid control subjects (n = 21). End-products of nitric oxide synthesis were measured from venous blood samples using nitrate reduction and chemiluminescence. To detect in vitro suppression of nitric oxide synthesis, human umbilical vein endothelial cell monolayers were grown to confluence and exposed to culture media containing 20% severe preeclamptic or control sera. Nitrate and nitrite production were compared in duplicate monolayers for each experimental condition, expressed as means +/- SEM in picomoles per 10(6) cells. Data were compared by Student's t or Mann-Whitney U tests, when appropriate, along with Spearman correlations for comparisons of laboratory and clinical data. RESULTS: Circulating nitrate and nitrite levels were similar in normotensive and preeclamptic cohorts (976 +/- 88 vs 1009 +/- 41 pmol/ml, respectively; p = 0.22), and no correlations between blood pressure and nitric oxide metabolite levels were observed for the control or severely preeclamptic subsets. Similar patterns of in vitro endothelial nitrite production were observed after 1-, 12-, and 24-hour incubations with 20% control or preeclamptic sera. CONCLUSIONS: Circulating nitrate and nitrite levels are not reduced in patients with severe preeclampsia compared with normotensive controls, and sera from these women do not suppress endothelial cell nitric oxide synthesis in vitro.

Endotoxin induces PAF production in the rat ileum: quantitation of tissue PAF by an improved method.

PAF (platelet-activating factor) is an endogenous mediator of endotoxin (LPS) shock and intestinal injury. In the present study we used an improved method to quantitate intestinal PAF after LPS injection. Both column and thin layer chromatography (TLC) were used to purify PAF. We found that using C18 column eluted sequentially with 10% acetic acid, ethyl acetate and 70% ethanol, yielded consistent results. TLC yielded falsely high PAF values, possibly from an unknown tissue lipid which co-migrated with PAF, or from toxic ingredients in the silica gel. Moreover, addition of optimal amounts of Tween-20 or ethanol in the bioassay samples enhanced PAF solubility and markedly improved PAF recovery. Lastly, dilution and heparinization of platelet-rich plasma greatly improved the sensitivity of the bioassay. The overall PAF recovery under these optimal conditions was 70-80%. We found that LPS (2-10 mg/kg, iv, 90 min) stimulated PAF production in the rat ileum, but not in the jejunum and colon. The difference in PAF production did not correlate to the numbers of sequestered neutrophils (reflected by myeloperoxidase levels) after LPS injection. This selective PAF production may account for the special vulnerability of the ileum to develop injury during endotoxemia.

Inducible platelet adherence to human umbilical vein endothelium by anticardiolipin antibody-positive sera.

OBJECTIVE: Our purpose was to determine whether anticardiolipin antibody-positive sera alter platelet adherence to vascular endothelium by a platelet activating factor-dependent mechanism. STUDY DESIGN: Anticardiolipin antibody-positive sera were used in an in vitro platelet-endothelial adherence assay. Confluent endothelial monolayers were randomly assigned for exposure to a 20% concentration of experimental and control sera. Platelets were radiolabeled with chromium 51, and adherence was assessed by quantification of endothelium-associated gamma emission. RESULTS: Inducible platelet adherence was observed by endothelial cell preincubation with sera from anticardiolipin antibody-positive donors compared with anticardiolipin antibody-negative control experiments (n = 12, platelet adherence 6.4% +/- 1.3% vs 4.5% +/- 1.1%, respectively; p = 0.02). Compared with endothelial cell incubations alone, coincubation of platelets with anticardiolipin antibody-positive sera consistently augmented primary adherence (n = 6, p = 0.042). Furthermore, platelet-adherence induced by antibody-positive sera was consistently attenuated by specific platelet-activating factor antagonists in a dose-dependent fashion (p < 0.001) and was restored by exogenously administered platelet-activating factor. CONCLUSIONS: Anticardiolipin antibody-induced platelet adherence may constitute an important prerequisite for vascular thrombosis in antibody-positive patients. The findings from this in vitro model suggest direct involvement of platelet-activating factor in this process.

Endogenous bacterial toxins are required for the injurious action of platelet-activating factor in rats.

BACKGROUND & AIMS: Platelet-activating factor (PAF), an endogenous mediator for experimental sepsis, has been shown to induce shock and intestinal necrosis in vivo. However, it is unclear whether PAF exerts its injurious effects on the intestinal tissue directly or via synergism with other endogenous products. The aim of this study was to examine the role of endogenous bacterial products, such as endotoxin, in PAF-induced intestinal injury. METHODS: PAF (3 micrograms/kg) was injected intravenously into normally colonized rats, germfree rats, and normal rats pretreated with a combination of antibiotics, and the systemic response and intestinal injury were assessed. RESULTS: PAF did not cause prolonged shock, leukopenia, hemoconcentration, and bowel necrosis in germfree rats. When germfree rats were primed with a low dose (0.5 mg/kg) of endotoxin, the protection was lost. Combined treatment of the normally colonized rats with neomycin, polymyxin B, and metronidazole for 7 days largely protected the animal from PAF-induced shock and intestinal necrosis. CONCLUSIONS: PAF does not directly induce prolonged hypotension, hemoconcentration, persistent leukopenia, and gross intestinal necrosis but causes these changes via a synergism with endogenous bacterial toxins, presumably from the gut flora.

An introduction to the structure and function of inflammatory mediators for clinicians.

As is apparent from a brief overview of some of the more important mediators involved in perinatal physiology and disease states, basic science research has provided many clinically relevant observations that, as discussed in other articles in this issue, have resulted in the discovery and development of clinically effective medications used daily in the care of the gravid mother and her fetus or neonate. In addition, an excellent base for the understanding of the mechanisms of the physiology of the maternal-fetal-placental unit has been established via extensive general and more focused research involving mediators.

Endotoxin-resistant mice are protected from PAF-induced bowel injury and death. Role of TNF, complement activation, and endogenous PAF production.

C3H/HeJ (endotoxin-resistant) mice have been used widely in biological research. However, the mechanism of endotoxin (LPS) resistance is only partially understood. In this study, we first investigated the differences in response to PAF, a mediator of endotoxin shock, between normal and C3H/HeJ mice. We found that in control mice, PAF (2.5 micrograms/kg) caused shock, hemoconcentration, complement activation, intestinal hypoperfusion, and necrosis with 75% mortality, whereas all C3H/HeJ mice survived, without complement activation or intestinal injury, and manifesting only mild hypotension and hemoconcentration. PAF also caused elevated serum TNF-alpha in some control mice but not in C3H/HeJ mice. We also observed that PAF induces endogenous PAF production and intestinal phospholipase A2 activation in normal mice, whereas PAF production and phospholipase A2 are suppressed in C3H/HeJ mice. The low endogenous PAF production may account, at least in part, for the resistance to LPS and PAF of C3H/HeJ mice.

Cardiopulmonary bypass with adequate flow and perfusion pressures prevents endotoxaemia and pathologic cytokine production.

Endotoxin and cytokine inflammatory mediators comprise the afferent and efferent limbs of the 'acute phase response'. During cardiopulmonary bypass (CPB) there may be gut translocation of endotoxin and contact activation of lymphocytes. It has been hypothesized that the haemodynamic instability encountered following CPB is caused by the 'acute phase response'. In this study we attempted to quantify the acute phase response in patients undergoing open-heart surgery and determine the influence of these cytokines on perioperative morbidity. Four perioperative blood samples were drawn from 20 consecutive patients undergoing coronary artery bypass grafting (CABG). These samples were assayed for endotoxin and four cytokines. In all cases the cardiac index was maintained > 2.4 l/min/m2 during nonpulsatile normothermic bypass (37 degrees C) and > 1.8 l/min/m2 during nonpulsatile hypothermic bypass (28 degrees C), and the perfusion pressure > 60 mmHg. Endotoxin was not detected in any of the test samples despite positive nonpatient controls. Interleukin 6 (IL-6) and tumour necrosis factor (TNF) were not detected despite an assay sensitivity of 80 and 10 pg/ml, respectively. TNF was detectable with an assay sensitivity of 0.5 pg/ml although there were no significant differences within the group. Interleukin 1 (IL-1) was detected (range = 0.98 - 9.09 ng/ml) in patients and again there were no trends within the group. The platelet activating factor (PAF) values peaked at crossclamp release (1.3 ng/ml versus a baseline of 0.2 ng/ml); however, there was no significant difference within the study.