Luba Adler

Role of Asphyxia and Feeding in a Neonatal Rat Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a common gastrointestinal disorder affecting premature infants. To investigate critically the importance of the purported risk factors of NEC (formula feeding, asphyxia, bacteria, and prematurity), we developed a neonatal rat model that closely mimics the human disease. Full-term and premature newborn rats were stressed with formula feeding, asphyxia, and/or exogenous bacterial colonization and subsequently evaluated grossly and histologically for the development of intestinal injury. We found that most animals treated with asphyxia, formula feeding, and bacteria developed NEC (77%) and died (86%) by 96 h. All maternally fed animals treated with asphyxia and bacterial colonization survived and had normal intestinal histology. Furthermore, asphyxia was a critical instigating factor, because formula and bacterial exposure without asphyxia resulted in normal intestine and minimal mortality (12%). Enteral bacterial colonization was not a significant determinant of NEC in this model. We conclude that the neonatal rat model is an excellent test system for the study of NEC. As in the human disease, asphyxia and formula feeding play an important role in the pathophysiology of experimental NEC.

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.

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 Students 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.

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.

Hypoxia increases stimulus-induced PAF production and release from human umbilical vein endothelial cells

Hypoxia alters endothelial cell function and metabolism. Since PAF is synthesized by endothelial cells and capable of modulating endothelial cell responses, we investigated the effect of hypoxia on synthesis and release of PAF from endothelial cells. We found: (1) Approx. 90% of the radylPAF derivative in stimulated endothelial cells is acylPAF. (2) Acute hypoxic (15 min-1 h) priming increased ionophore- and thrombin-induced radylPAF accumulation. (3) Long-term hypoxic exposure increased radylPAF accumulation at 24 and 48 h in the presence of ionophore. (4) Bioactive PAF was released into media and hypoxia and ionophore synergistically increased PAF release. (5) Hypoxia and ionophore stimulation increased phospholipase A2 activity and decreased acetylhydrolase activity in endothelial cells. We conclude that hypoxia and ionophore increase PAF synthesis and release from endothelial cells.

Anticardiolipin antibody -positive serum enhances endothelial cell platelet-activating factor production

Circulating antiphospholipids have been linked to recurrent pregnancy loss by a mechanism involving placental and decidual thrombosis. We hypothesized that platelet-activating factor, an autacoid synthesized by vascular endothelium, might mediate this phenomenon through its ability to promote platelet aggregation and fibrin deposition. Alternatively, antiphospholipid antibodies might exert a procoagulant effect by inhibiting the synthesis of prostacyclin. To evaluate these theories, endothelial cells (harvested from human umbilical veins) were grown to confluence and incubated for 48 hours with 20% concentrations of anticardiolipin antibody-positive and -negative human sera as well as fetal bovine serum. After incubation culture wells were stimulated with 10 mumol/ml calcium ionophore A23187 (an agonist of platelet-activating factor and prostacyclin synthesis). Intracellular platelet-activating factor was measured by tritiated acetate incorporation, phospholipid extraction, thin-layer chromatography, and scintillation spectrophotometry. Enhanced platelet-activating factor synthesis was identified in cultures incubated with anticardiolipin antibody-positive serum (25,544 +/- 2604 disintegrations per minute, mean +/- SD) when compared with anticardiolipin antibody-negative serum (18,600 +/- 3316 dpm) or fetal bovine serum (19,014 +/- 4233 dpm; analysis of variance, p = 0.033). In similar experiments, prostacyclin synthesis was determined by measuring its primary metabolite, 6-keto-prostaglandin F1 alpha, in culture supernatants. No differences between anticardiolipin antibody-positive and control cultures were observed (analysis of variance, p = 0.90). We conclude that in this endothelial cell model, anticardiolipin antibody-positive serum enhances ionophore-mediated platelet-activating factor synthesis but has no apparent effect on the production of prostacyclin. These findings suggest a potential role for platelet-activating factor in anticardiolipin antibody-mediated vascular thrombosis.

Evaluation of a leukotriene receptor antagonist in prevention of hyperoxic lung injury in newborn rabbits

Prolonged exposure to hyperoxia can result in significant lung injury and has been associated with the development of bronchopulmonary dysplasia. Leukotrienes (LT) recruit polymorphonuclear leukocytes (PMN) to the lung, increase vascular permeability, and have therefore been postulated to play a role in the pathogenesis of hyperoxic lung injury. This study investigates ICI 198,615 (ICI), an LTD4 and LTE4 receptor antagonist in preventing hyperoxic lung injury in newborn rabbits. Matched littermates of 7-day-old rabbits received ICI (0.1 or 1.0 microM/kg/h) or vehicle alone, were exposed to greater than 95% O2, and sacrificed after 48, 72, 84 and 96 h of exposure. Bronchoalveolar alveolar lavage fluid (BAL) of the left lung was analyzed for white cell count, differential, absolute number of PMNs, total protein, and cyclooxygenase products 6-keto-PGF1 alpha, and thromboxane B2. Lung water was quantified utilizing the right lung. Results demonstrated no significant differences between the ICI groups or between the ICI groups and controls. In conclusion, the administration of the LTD4 and LTE4 receptor antagonist ICI 198,615 was insufficient to reduce the formation of pulmonary edema, reduce mortality or attenuate hyperoxic lung injury. These experiments suggest that a number of other mediators may be involved in the hyperoxic lung injury process and that the functional inhibition of a portion of the arachidonic acid cascade was not sufficient to either prevent or attenuate hyperoxic lung injury in newborn rabbits.

Role of ecosanoids in relative oxygen tolerance of newborn rabbits

Prolonged exposure to hyperoxia can result in significant lung injury, although newborn animals are more oxygen-tolerant than adults. Mechanisms affording tolerance to the newborn are incompletely understood. This study examined the hypothesis that eicosanoids play a significant role in newborn oxygen tolerance. One litter of term newborn albino rabbits and 15 adult rabbits were exposed to 65 hours of greater than 95% O2. An additional litter of newborns served as a normoxic control. Normoxic newborn rabbits had very high quantities of 6-keto-PGF1a and low TXB2 in bronchoalveolar lavage (BAL) fluid. Sixty-five hours of oxygen exposure in newborn rabbits produced no evidence of lung injury on light microscopy, 97% of BAL white cells were alveolar macrophages and BAL protein was low. An equal period of oxygen exposure produced significant lung injury in adult rabbits. BAL fluid from oxygen-injured adults contained a 17-fold greater percentage of PMN and 16-fold higher protein than oxygen-exposed newborns. Hyperoxic adults had significantly lower 6-keto-PGF1a, and significantly higher LTB4 and LTC4 in BAL compared to hyperoxic newborns. This study confirms the hypothesis of relative oxygen tolerance in newborn rabbits compared to adults, and suggests that this tolerance may have been afforded by higher pulmonary levels of the protective prostacyclin metabolite.

Platelet Activating Factor Alters Ion Transport and Barrier Function in Intestinal Epithelial Cells

Platelet Activating Factor Alters Ion Transport and Barrier Function in Intestinal Epithelial Cells

Endotoxin Increases Type II-Phospholipase A 2 Gene Expression in Rat Intestinal Epithelial Cells

Endotoxin Increases Type II-Phospholipase A 2 Gene Expression in Rat Intestinal Epithelial Cells