Objective
Maternal infection is associated with oxidative stress and inflammation. We sought to determine whether N-acetyl-cysteine can decrease maternal oxidative stress and the inflammatory response in preterm gestation.
Study Design
Pregnant rats 16 days, were treated with (1) lipopolysaccharide, (2) N-acetyl-cysteine 120 minutes after lipopolysaccharide, or (3) saline solution (intraperitoneal). Six hours after lipopolysaccharide administration, serum lipid peroxide formation (LPO), tumor necrosis factor-α, interleukin-6, and interleukin-1β levels in maternal serum and amniotic fluid were determined.
Results
Lipopolysaccharide significantly increased maternal serum lipid peroxide formation (24-118.5 nmol/mL; P < .05), and maternal serum and amniotic fluid tumor necrosis factor-α, interleukin-6, and interleukin-1β. N-acetyl-cysteine treatment after lipopolysaccharide significantly attenuated lipid peroxide formation (47.5 nmol/mL) and proinflammatory cytokines response in maternal serum and amniotic fluid.
Conclusion
Maternal and amniotic fluid oxidative stress and inflammatory stimulation are attenuated by N-acetyl-cysteine even when administered after lipopolysaccharide. These results suggest that N-acetyl-cysteine may protect the fetus from adverse sequelae associated with inflammatory stimulation.
Increased maternal oxidative stress in pregnancy is associated with an increased risk of poor pregnancy outcome. Periventricular-intraventricular hemorrhage, necrotizing enterocolitis, chronic lung disease, and retinopathy of prematurity have been referred to as oxygen radical diseases as they are related to excess oxidant stress relative to antioxidant defenses in premature infants.
Cerebral palsy (CP) is associated with intrauterine inflammation, preterm birth, and low birthweight. Most recently, fetal brain injury and offspring CP has been associated with maternal infection/inflammation. Several studies have demonstrated that maternal urinary tract infection increases the risk of CP from 4- to 6-fold, among preterm or term infants, though not all studies demonstrate this association. Systemic maternal infections, as evidenced by maternal fever are also linked to offspring CP.
Several mechanisms have been proposed for maternally induced fetal inflammation and injury, lipopolysaccharide (LPS), derived from the cell wall of Gram-negative bacteria can directly induce proinflammatory (including interleukin-6 [IL-6] and tumor necrosis factor-α [TNF-α],) cytokines, which function to drive the inflammatory response. Prolonged activation of proinflammatory responses have been implicated in white matter damage and is associated with astrogliosis and a loss of prooligodendrocytes. LPS may also induce mediators of oxidative stress, which themselves induce cytokine production. Oxidative stress leads to cytokine induction via activation of nuclear factor-κB (NF-κB), which is a major transcription factor for TNF-α, IL-6, and IL-8.
As cytokine responses may be mediated by oxidative stress, and the preterm fetus is uniquely vulnerable to neurologic damage, we sought to determine whether changing the reduction-oxidation balance by enhancing the activity or the availability of antioxidants may modulate the inflammatory response during pregnancy, thereby reducing the risk to the developing fetus. In this study, we have sought to determine the effects of N-acetyl-cysteine (NAC), an antioxidant shown to inhibit NF-κB activity in culture, on the induction of oxidative stress and cytokines in maternal serum and amniotic fluid in response to LPS in early preterm pregnancy.
Materials and Methods
Animals and treatments
Sprague-Dawley pregnant rats (Harlan Sprague Dawley, Inc, Madison, WI) were obtained at gestational day 14 (day 1 is sperm plug observed, term = 21) and allowed to acclimate for 48 hours before the beginning of experiments. Animals were maintained in temperature (37°C) and light (0600-lights on; 1800-lights off) controlled facilities with access to food (LabDiet 5001 Rodent Diet; PMI Nutrition International, LLC, St. Paul, MN) and water ad libitum throughout the study. Administration of saline solution, NAC, or LPS was done intraperitoneally. NAC (Sigma-Aldrich, St. Louis, MO) was reconstituted in physiologic saline solution at pH of 6.8-7.2 (adjusted with sodium hydroxide [NaOH], and administered at 300 mg/kg birthweight). LPS ( Escherichia coli , serotype 0111:B4; Calbiochem, La Jolla, CA) was reconstituted in physiologic saline solution and administered at 100 μg/kg birthweight.
At gestational day 16, the pregnant rats received injections of physiologic saline solution (Sal) or LPS at time 0, followed by saline solution or NAC (300 mg/kg birthweight [Sigma-Aldrich]) at time 120 minutes (Sal-Sal, LPS-Sal, LPS-NAC). There were 5 pregnant rats in the Sal-Sal group, 6 pregnant rats in the LPS-Sal group, and 6 pregnant rats in the LPS-NAC group. The protocols and procedures for this study were approved by the Institutional Animal Care and Utilization Committee (IACUC) at Rappaport Research and Education Institute.
Sample collection
Six hours after LPS or saline solution injections, pregnant rats were anesthetized with pentobarbital (intramuscular) and the heart and peritoneal cavities were exposed via midline incision. Maternal blood was collected via cardiac puncture and centrifuged at 4°C to isolate serum. The uterus was removed and placed in a chilled Petri dish. Each amniotic sac was punctured and amniotic fluid was pooled from all the fetuses of the same rat and collected into microcentrifuge tubes and quickly frozen in liquid nitrogen. All samples were subsequently stored at −80°C for further processing and analyses. All samples were individually analyzed.
Serum lipid peroxidation
Serum was diluted 1:4 (v:v) with phosphate buffered saline solution (PBS) and then incubated for 2 hours at 37°C. Serum lipid peroxidation was determined by measuring the generated amount of lipid peroxides (PD) using spectrophotometric methods. The PD test analyzes lipid peroxide formation by their capacity to convert iodide to iodine measured spectrophotometrically at 365 nm.
Enzyme-linked immunoabsorbant assay determinations
Commercial enzyme-linked immunoabsorbant assays (ELISA, R&D Systems, Minneapolis, MN) kits were used to determine maternal serum and amniotic fluid protein levels of the cytokines IL-6, IL-1β, and TNF-α as previously described. All samples were measured in duplicates and mean values used. For all assays, the minimum detectable dose was <10 pg/mL with intra- and interassay variations <10%.
Data analyses
Blood and amniotic fluid levels of inflammatory biomarkers were compared between the different groups. As maternal and amniotic responses were not normally distributed, values were analyzed using Kruskal-Wallis nonparametric tests and Dunn’s post test. All results are expressed as median and interquartile range and means ± standard error of measurement (SEM). Difference was considered to be significant only for P < .05.
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