Maternal N-acetyl-cysteine prevents neonatal brain injury associated with necrotizing enterocolitis in a rat model.

INTRODUCTION: Preterm infants with necrotizing enterocolitis (NEC) are at increased risk of cerebral injury and neurodevelopmental dysfunction. N-acetyl-cysteine (NAC) is a known anti-inflammatory and antioxidant agent. Currently, there is no prophylactic treatment in clinical use to prevent NEC and its neurodevelopmental sequelae. We sought to determine whether brain inflammation/apoptosis accompanies NEC systemic inflammation, and whether it can be attenuated by maternal NAC treatment during pregnancy and/or in the neonatal period in a rat model. MATERIAL AND METHODS: An established NEC newborn model (hypoxia 5% O2 for 10 min and formula feeding thrice daily, beginning on day 1 for 4 days) was used in Sprague-Dawley rat pups (n = 32). An additional group of pups (n = 33) received NAC (300 mg/kg intraperitoneal thrice daily) in addition to NEC conditions (NEC-NAC). Control pups (n = 33) were nursed and remained with the dam in room air. Two additional groups included pups of dams treated once daily with NAC (300 mg/kg intravenous) in the last 3 days of pregnancy. After birth, pups were randomized into NAC-NEC (n = 33) with NEC conditions and NAC-NEC-NAC (n = 36) with additional postnatal NAC treatment. Pups were sacrificed on the fifth day of life. Pup serum interleukin (IL)-6 protein levels, and brain nuclear factor kappa B (NF-κB) p65, neuronal nitric oxide synthase (nNOS), Caspase 3, tumor necrosis factor alpha (TNF-α), IL-6 and IL-1ß protein levels were determined by ELISA, western blot and TUNEL staining, and the groups were compared using analysis of variance (ANOVA). RESULTS: NEC pups had significantly increased serum IL-6 levels compared with the control group as well as increased neuronal apoptosis and brain protein levels of NF-κB, nNOS, Caspase 3, TNF-α, IL-6 and IL-1ß compared with control. In all NAC treatment groups, levels of serum IL-6, neuronal apoptosis and brain NF-κB, nNOS, Caspase 3, TNF-α, IL-6 and IL-1ß protein levels were significantly reduced compared with the NEC group. The most pronounced decrease was demonstrated within the NAC-NEC-NAC group. CONCLUSIONS: NAC treatment can attenuate newborn inflammatory response syndrome and decrease offspring brain neuroapoptosis and inflammation in a rat model of NEC by inhibition of NF-κB, nNOS and Caspase 3 pathways.

Prophylactic antenatal N-Acetyl Cysteine administration combined with postnatal administration can decrease mortality and injury markers associated with necrotizing enterocolitis in a rat model.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of neonates, especially premature neonates. To date, there is no prophylactic treatment against NEC, except breast milk and slow increase in enteral feeding, and there is no antenatal prophylaxis. AIMS: To assess possible protective effects of antenatal N-Acetyl Cysteine (NAC) against the intestinal pathophysiological changes associated with NEC in a rat model of NEC and against its associated mortality. METHODS: Newborn Sprague-Dawley rats were divided into 5 groups: control (n = 33); NEC (n = 32)-subjected to hypoxia and formula feeding for 4 days to induce NEC; NEC-NAC (n = 34)-with induced NEC and concomitant postnatal NAC administration; NAC-NEC (n = 33)-born to dams treated with NAC for the last 3 days of pregnancy starting at gestational age of 18 days, and then subjected to induced NEC after birth; NAC-NEC-NAC (n = 36)-subjected to induced NEC with both prenatal and postnatal NAC treatment. At day of life 5, weight and survival of pups in the different groups were examined, and pups were euthanized. Ileal TNF-α, IL-6, IL-1ß, IL-10, NFkB p65, iNOS and cleaved caspase 3 protein levels (western blot) and mRNA expression (RT-PCR) were compared between groups. RESULTS: Pup mortality was significantly reduced in the NAC-NEC-NAC group compared to NEC (11% vs. 34%, P<0.05). Ileal protein levels and mRNA expression of all injury markers tested except IL-10 were significantly increased in NEC compared to control. These markers were significantly reduced in all NAC treatment groups (NEC-NAC, NAC-NEC, and NAC-NEC-NAC) compared to NEC. The most pronounced decrease was observed in the NAC-NEC NAC group. CONCLUSIONS: Antenatal NAC decreases injury markers and mortality associated with NEC in a rat model. Antenatal administration of NAC may present a novel approach for NEC prophylaxis in pregnancies with risk for preterm birth.

Fetal neuroprotective mechanism of maternal magnesium sulfate for late gestation inflammation: in a rodent model.

Background: Maternal administration of magnesium sulfate (Mg) is used in humans to protect the fetal brain during preterm delivery. We sought to determine the neuroprotective mechanism of Mg in a rat model of late gestation maternal inflammation.Methods: Pregnant rats at 20 d of gestation (20 total, four groups, N = 5 in each group) received i.p. LPS or saline. Dams were randomized for s.c. saline or Mg supplementation 2 h prior and following the LPS/saline injections. Dams were sacrificed 4 h following the last treatment. Fetal brains were collected from the four treatment groups. Fetal brain caspase 3 active form, NF-kB p65, neuronal nitric oxide synthase (phospho-nNos), and proinflammatory cytokines levels were determined by western blot.Results: Maternal LPS at e20 significantly (p < .01) increased fetal brain caspase 3 active form (af) (0.27 ± 0.02 versus 0.15 ± 0.06u), NFkB (0.23 ± 0.01 versus 0.13 ± 0.01u), and phospho-nNOS (0.22 ± 0.01 versus 0.12 ± 0.01u) and fetal brain proinflammatory cytokines (IL-6 0.21 ± 0.01 versus 0.11 ± 0.01 u; TNFα 0.29 ± 0.01 versus 0.15 ± 0.01u), compared with control fetuses. Mg treatment significantly (p < .05) reduced fetal brain caspase 3 af (0.16 ± 0.01u), NFkB p65 (0.11 ± 0.01u), phospho-nNOS (0.1 ± 0.01u), as well as brain proinflammatory cytokines (IL-6 0.07 ± 0.01u; TNFα 0.15 ± 0.01u) to levels similar to controls.Conclusion: Maternal inflammation-induced fetal brain injury at late gestation may be mediated by the activation of inflammatory response, oxidative stress, and apoptosis. Maternal Mg may attenuate the injury by inhibition of these putative pathways.