Intrauterine Lipopolysaccharide Research Articles

N-acetylcysteine attenuates the maternal and fetal proinflammatory response to intrauterine LPS injection in an animal model for preterm birth and brain injury

Objective. Maternal immune activation (MIA) is associated with preterm birth (PTB) and abnormal neurologic outcome. We hypothesized that N-acetylcysteine (NAC) would decrease PTB and neonatal brain injury acting as an anti-inflammatory.Methods. Pregnant CD-1 mice received intrauterine LPS or saline on day 15/20. They received NAC or saline and were monitored until delivery. Pups were followed and sacrificed on postnatal days 1/30 and brains were collected. Immunostaining for heavy-chain neurofilament protein (NF-H), myelin basic protein (MBP), and proteolipid protein (PLP) was performed. In another group, animals were sacrificed 6 h after treatment, and fetal brain, placenta, and myometrium were collected. Il-6, Il-1β, Il-10, and tumor necrosis factor (TNF)-α mRNA expression was determined. Nonparametric analysis was used for analysis, and pairwise comparisons were performed when appropriate.Results. Lipopolysaccharide (LPS) caused PTB (79 vs. 0%, p < 0.005), and this was reduced by NAC [0.45 (95% CI: 0.26–0.83), p < 0.008]. LPS increased IL-6 expression in myometrium and placenta. This was attenuated by NAC in myometrium. IL-1β, IL-6, and TNF-α expression increased in the fetal brain with LPS. LPS produced altered NF-H, MBP, and PLP staining, and these effects were attenuated by NAC.Conclusion. NAC attenuates inflammation in this MIA model and reduces PTB and white matter injury. It is an interesting candidate for study for prevention of PTB and neurologic injury.

Expression of myelin basic protein, glial fibrillary acidic protein, interleukin-1β and tumor necrosis factor-α in neonatal rats brain after intrauterine exposure on lipopolysaccharide

Objective To probe into the expression of inflammatory factrors and white matter damage after intrauterine lipopolysaccharide(LPS) exposure in neonatal rats brain. Methods LPS (0. 4 μg) was administrated into the intrauterine cavity between every two embryo sacs of SD rats at day 19 of gestation in LPS group(n= 14) , while pyrogen-free saline was administrated in control group (n=10). Neonatal brain tissues were collected at postnatal day 1,3,7 and 11. Reverse transcription-polymerase chain reaction analysis was used to examine mRNA expression of interleukin-1βand tumor necrosis factor-a, and immunohistochemistry was used to evaluate the expression of myelin basic protein and glial fibrillary acidic protein in neonatal brain tissues. Statistical analysis was performed using t test. Results Placental hematoxylin-eosin staining in the LPS group showed distinct inflammatory cell infiltration, prominent hyperplasia of interstitial tissue and narrowed capillary. Myelin basic protein in cerebellum and cerebrum of neonatal rats at postnatal 11 days in LPS group was much weaker than in control group (cerebellum: 1. 29 ±0. 76 vs 2. 43 ±0.79, t =2. 038,P=0.045; cerebrum: 1. 71 ±0. 49 vs 2.50±0.76, t= 2. 420,P = 0.032). Glial fibrillary acidic protein of neonatal rats brain at postnatal 11 days in LPS group was much stronger than in control group (hippocampus: 2.71±0.49 vs 1.75±0.50, (t=-3.029, P = 0.026. corpus callus: 2.40±0.55 vs 1. 50±0. 58, t= -2. 646,P = 0. 019. cerebrum: 1. 50±0. 55 vs 1. 00±0. 00, t= -2. 236, P=0. 049. cerebellum: 2. 80 ±0. 45 vs 1.60 ±0.55, t = -3.08,P =0.009). But there was no statistical difference between LPS group and control group in the expressions of IL-1β and TNF-α mRNA in neonatal rats brain at each time point. Conclusions Intrauterine LPS exposure may decrease the placental blood flow resulting in neonatal low birth weight and white matter damage, which characterized by astrogliosis and hypomyelination. Key words: Pregnancy complications, infectious) Lipopolysaccharides; Animals, newborn

The Cyclopentenone 15-Deoxy-Δ12,14-Prostaglandin J2 Delays Lipopolysaccharide-Induced Preterm Delivery and Reduces Mortality in the Newborn Mouse

Intrauterine infection is a common trigger for preterm birth and is also a risk factor for the subsequent development of neurodevelopmental abnormalities in the neonate. Bacterial lipopolysaccharide (LPS) binds to toll-like receptor-4 (TLR-4) to activate proinflammatory signaling pathways, which are implicated in both preterm delivery and antenatal brain injury. The transcription factor nuclear factor-kappaB (NF-kappaB) is a key player in the orchestration of the inflammatory response and has a central role in parturition. Here we show that intrauterine administration of TLR-4-specific LPS to pregnant mice results in the activation of NF-kappaB in the maternal uterus and the fetal brain, up-regulation of proinflammatory proteins cyclooxygenase-2, chemokine ligand 1, ChemoKine (C-C motif) ligand 2, and cytosolic phospholipase A(2) in myometrium, and induction of preterm delivery. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is an antiinflammatory prostaglandin that plays a role in promoting the resolution of inflammation. We report that coadministration of 15d-PGJ(2) and LPS to pregnant mice delays LPS-induced preterm delivery and confers protection from LPS-induced fetal mortality. This is associated with inhibition of myometrial NF-kappaB, cytosolic phospholipase A(2), and c-Jun N-terminal kinase activation, and of inflammatory protein synthesis. Therefore 15d-PGJ(2) has anti-inflammatory effects via inhibition of multiple aspects of inflammation-driven TRL-4 signaling pathway. Thus, 15d-PGJ(2) or compounds with similar antiinflammatory functions may have potential as therapeutic agents in the management of preterm labor with the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation.

Modulation of Monocyte Chemotactic Protein-1 Expression During Lipopolysaccharide-Induced Preterm Delivery in the Pregnant Mouse

Preterm delivery is often associated with increased cytokine and chemokine production. These studies characterize the expression of the chemokine monocyte chemotactic protein-1 (MCP-1) in mice during lipopolysaccharide (LPS)-induced preterm delivery. Uterine and other tissues were harvested from CD-1 mice on gestational day 15 after intrauterine LPS injection. Quantitative real-time reverse-transcriptase polymerase chain reactions determined MCP-1 and toll-like receptor 4 (TLR4) mRNA expression during the 24 hours after LPS. MCP-1 protein expression was determined using a cytokine/chemokine protein array, enzyme-linked immunosorbant assay, and immunohistochemistry. Intrauterine LPS injection caused preterm delivery in CD-1 mice between 12 and 24 hours. Expression of MCP-1 mRNA significantly increased at 2 and 6 hours, while TLR4 expression did not significantly change over 24 hours. The MCP-1 protein levels peaked by 2 to 6 hours in maternal serum, liver, lung, kidney, and uterus. Immunohistochemistry confirmed MCP-1 in the myometrium and endometrium. These studies provide evidence suggesting that MCP-1 potentially plays an important role during the proinflammatory immune response, leading to preterm labor in the mouse.

Effects of intrauterine inflammation on the developing mouse brain

Clinical and experimental evidence indicate that the presence of intrauterine inflammation in pregnancy is not only a cause of preterm birth but is also associated with perinatal brain damage and long-term neurological handicap. In the present study, the neuropathological outcome was investigated in surviving pups in a model of inflammation-induced preterm delivery. C57BL/6 mice were subjected to intrauterine injection of lipopolysaccharide (LPS) or saline, at a time corresponding to 79% of average gestation (gestational day 15). Fetuses that survived after LPS administration were sacrificed on postnatal day 14 (PND 14). At PND 14, the brain weight of LPS-exposed pups was significantly lower than that of saline-exposed. A high proportion of LPS-exposed brains were found affected and exhibited hypomyelination, enlarged ventricles, and in some cortical gray matter lesions were evident. None of these pathologies were detected in sham-treated animals. Conclusions: Intrauterine inflammation impaired brain development and various brain lesions were produced in both the white and gray matter after intrauterine LPS administration in mice.

The use of progestational agents for preterm birth: Lessons from a mouse model

On the basis of the recent Maternal Fetal Medicine Unit Networks clinical trial, the American College of Obstetricians and Gynecologists supports the administration of 17-alpha hydroxyprogesterone caproate to high-risk patients. Because inflammation/infection is believed to be a contributing factor in many cases of preterm birth, it is imperative to understand the effect of 17-alpha hydroxyprogesterone caproate treatment in this clinical situation. Using a mouse model of localized intrauterine inflammation, we investigated the ability of progestational agents to prevent preterm birth. On gestational day 15 (E15), dams were assigned randomly to treatment with 17-alpha hydroxyprogesterone caproate, medroxyprogesterone acetate, or vehicle before intrauterine infusion of lipopolysaccharide. All dams were monitored for morbidity and preterm birth. Three separate sets of experiments were performed to assess different outcomes at 6, 24, and 96 hours. At 6 and 24 hours, C-reactive protein, interleukin-6, and interleukin-10 levels were measured in maternal serum by enzyme-linked immunosorbent assay. Pretreatment with 17-alpha hydroxyprogesterone caproate or medroxyprogesterone acetate before intrauterine lipopolysaccharide treatment significantly decreased the preterm birth rate, compared with lipopolysaccharide treatment alone. Medroxyprogesterone acetate treatment was more effective than 17-alpha hydroxyprogesterone caproate treatment in the prevention of preterm birth and resulted in live pups at term. Treatment with 17-alpha hydroxyprogesterone caproate was associated with significant maternal morbidity. In the setting of intrauterine inflammation, progestational agents decrease the preterm birth rate but can result in maternal morbidity. 17-Alpha hydroxyprogesterone caproate should not be used in patients who are suspected of having subclinical infection and/or acute preterm labor. The mechanisms by which progestational agents inhibit preterm birth warrants further investigations so that the use of this drug to appropriate populations could be pursued without undue fetal or maternal harm.

Disruption of Interleukin-18, but Not Interleukin-1, Increases Vulnerability to Preterm Delivery and Fetal Mortality after Intrauterine Inflammation

Preterm birth is a major contributor of adverse perinatal outcome. Clinical data suggest that an inflammatory response is important in the process leading to preterm labor. By using a recently introduced mouse model of localized intrauterine lipopolysaccharide-induced inflammation, the effect of interleukin (IL)-18 gene disruption and/or IL-18 neutralization as well as combined IL-1alpha/beta gene disruption on inflammation-induced fetal loss was investigated. The frequency of preterm fetal loss was significantly higher in IL-18 knockout mice (58.9%) and in mice administered IL-18-binding protein (59.7%) compared to wild-type controls (34.7%). The rate of fetal loss was not affected by IL-1alpha/beta gene deficiency (38.7%). Decreased IL-18 protein expression combined with elevated IL-12 protein expression in uterine tissue of IL-18 knockout mice and IL-18-binding protein-treated animals was noticed. These data demonstrate that preterm pregnancy loss in response to intrauterine inflammation was enhanced by disruption of the IL-18 gene and/or IL-18 neutralization, events that may relate to exaggerated Th1 responses because of an increased IL-12/IL-18 ratio.

Lipopolysaccharide-induced inflammation and perinatal brain injury

Both energy failure and infections are important risk factors for brain injury in term and preterm infants. In this review we focus on recent experimental studies that have examined the effects of lipopolysaccharide (LPS) exposure to the fetus or neonate and the interaction of LPS with other events. Intracerebral LPS injections induce a marked cerebral cytokine response and prominent white matter lesions. LPS administered intravenously to the fetus also induces gross lesions, which are mainly localised to the white matter and are accompanied by activation of inflammatory cells. Cerebral effects following fetal LPS exposure via more distant routes, such as intracervical, intrauterine or maternal LPS administration, are characterised by reductions in oligodendrocyte or myelin markers without macroscopic lesions being evident. Both antenatal and neonatal LPS exposures increase the sensitivity of the brain to subsequent hypoxic/ischaemic events, even in adulthood. These studies suggest that fetal inflammation is the strongest predictor of brain lesions.

Can medroxyprogesterone acetate alter Toll-like receptor expression in a mouse model of intrauterine inflammation?

Activation of the innate immune receptors, Toll-like receptors 2 and 4, are critical for a host inflammatory response to both Gram-positive and Gram-negative organisms. These receptors can initiate and modulate the inflammatory response. Differential regulation of Toll-like receptors may be one of the mechanisms by which intrauterine inflammation signals parturition. Likewise, progestational agents may have the ability to modify this effect. These studies were performed to elucidate the effect of intrauterine inflammation and medroxyprogesterone acetate on Toll-like receptor expression in the uterus, cervix, and placenta in a mouse model of intrauterine inflammation. On day 15 of gestation, CD-1 mice were randomized to pretreatment with medroxyprogesterone acetate or vehicle before intrauterine infusion with lipopolysaccharide or sterile saline solution. Six hours after intrauterine infusion, uterine, cervical, and placental tissues were harvested. RNA and protein were extracted. Quantitative polymerase chain reaction was performed for Toll-like receptor 2 and 4 messenger RNA. Western blot analysis was performed with Toll-like receptor 4-specific antibodies. Intrauterine inflammation up-regulated Toll-like receptor 2 and 4 messenger RNA in uterus, cervix, and placenta. Pretreatment with medroxyprogesterone acetate decreased the lipopolysaccharide-induced up-regulation of Toll-like receptor 2 and 4 messenger RNA in the cervix and placenta. Medroxyprogesterone acetate treatment, in the presence of lipopolysaccharide, was unable to prevent the lipopolysaccharide-induced increase in Toll-like receptor 4 messenger RNA and protein in the uterus. Medroxyprogesterone acetate treatment alone in pregnant mice significantly increased Toll-like receptor 4 messenger RNA expression in the uterus. Intrauterine inflammation has a differential effect on Toll-like receptor 2 and 4 expression. The observed up-regulation of Toll-like receptor 2 in the uterus in response to intrauterine lipopolysaccharide may be a mechanism to augment the inflammatory response and may serve to promote parturition in the setting of inflammation. Consequently, the ability of medroxyprogesterone acetate to suppress lipopolysaccharide-induced up-regulation of Toll-like receptor 2 messenger RNA may be one of the mechanisms by which progestins are able to decrease preterm birth.

Medroxyprogesterone acetate, but not progesterone, protects against inflammation-induced parturition and intrauterine fetal demise

Objective This study was undertaken to determine whether progestational agents can prevent inflammation-induced preterm parturition and fetal demise. Study design The activation of contractile and inflammatory pathways in response to localized intrauterine inflammation was investigated by using quantitative polymerase chain reaction (PCR). Serum progesterone (P4) levels and alterations in progesterone receptor-B (PR-B) were determined with radioimmunoassay and quantitative PCR, respectively. With our in vivo model of intrauterine inflammation, animals were randomly assigned to pretreatment with P4 or medroxyprogesterone acetate (MPA) before intrauterine lipopolysaccharide (LPS). Animals were observed for preterm delivery. The number of live pups 48 hours after intrauterine LPS was recorded for each treatment group. The ability of MPA to alter signal transduction pathways leading to preterm parturition were investigated by quantitative PCR and histochemical studies. Results Intrauterine inflammation is associated with decreased serum progesterone levels and decreased transcription of PR-B. Preterm delivery rates were 100% for LPS alone, 63% for LPS + P4, and 0% for LPS + MPA. No live pups remained at 48 hours in the LPS or LPS + P4 groups. Pretreatment with MPA significantly preserved fetal viability. MPA suppressed activation of contraction-associated genes and inflammatory mediators and prevented cervical ripening in response to intrauterine inflammation. Conclusion MPA, with its progestational and anti-inflammatory properties, prevented inflammation-induced preterm parturition and significantly preserved fetal viability.

Mechanisms of lipopolysaccharide-induced changes in effects of contractile agonists on pregnant rat myometrium

ObjectiveThe study was undertaken to investigate the mechanisms underlying enhanced uterine contractility induced by lipopolysaccharide (LPS) in pregnant rats. Study designWistar rats were administered intrauterine either LPS (50 μg) or normal saline solution (0.05 mL) on day 17 of gestation. On day 19, the animals were killed and uterus was isolated for isometric recording, 45Ca++ influx measurement, and determination of plasma membrane Na+-K+-ATPase. ResultsUterine strips, taken from LPS-treated rats, displayed a marked increase in amplitude of spontaneous rhythmic contractions compared with controls. Enhancement in the sensitivity of uterine strips to agonists such as oxytocin, 5-hydroxytryptamine (5-HT), and BAY K8644 was also observed in rats treated with LPS. Cyclo-oxygenase-2 inhibitor, nimesulide (10 μmol/L) had no significant effect on the LPS-induced increase in spontaneous rhythmic contractions. On the other hand, nimesulide attenuated the increased sensitivity of uterine strips to oxytocin induced by LPS. Nimesulide significantly inhibited 5-HT–induced uterine contractions in both control and LPS-treated rats. However, the enhanced sensitivity of uterine strips to 5-HT was evident even in the presence of nimesulide in rats treated with LPS. Nifedipine-sensitive 45Ca++-influx into uterine strips both in the basal state as well as those stimulated by high K+ (80 mmol/L) and 5-HT (1 μmol/L) was greater in LPS-treated group compared with the controls. LPS treatment caused a marked inhibition in the Na+-K+-ATPase activity of the uterine plasma membrane compared with controls. LPS had no effect on plasma 17β-estradiol levels. ConclusionLPS appears to increase uterine contractility of pregnant rats both through the release of endogenous prostaglandins and increased influx of Ca++ through L-type Ca++ channels. Inhibition of sodium pump by LPS may be an additional mechanism in augmentation of uterine excitability.

Intrauterine infusion of bacterial lipopolysaccharide (LPS) prior to mating has no adverse effect on fertility, fetal survival and fetal development

Lipopolysaccharide (LPS, endotoxin) is a component of the cell wall of gram-negative bacteria and a potent inducer of severe inflammatory reactions. In mice, systemically administered LPS induces fetal resorption and increases fetal mortality. However, effects of intrauterine LPS on fertility, fetal survival and development have not been reported. In the present study, pigs were used to determine the effect of intrauterine infused LPS on fertility, fetal survival and development. Prior to mating, gilts received intrauterine infusion of either a single dose of saline or increasing doses of LPS in saline using an insemination catheter. On day 30 of pregnancy, gilts were hysterectomized and litter size, fetal length, number of corpora lutea (CL), ovarian and placental weights, and allantoic and amniotic fluid volumes were recorded. Blood progesterone levels from days 10–30 of pregnancy were also determined. Results indicated that intrauterine infusion of LPS had no adverse effects on blood progesterone levels, fertility, fetal survival or fetal development. Intrauterine injection of LPS did cause an increase in fetal weight and amniotic fluid volume ( P<0.05). These results suggest that sperm, oocytes and gametes are tolerant of local LPS challenge and, to some extent, this mechanism protects gametes and conceptuses from maternal response to mating-introduced bacteria and their potential endotoxins.