Study On Dendrimer-Based Nac Therapy For Preterm Birth And Perinatal Brain Injury Induced By Intrauterine Inflammation In A Pregnant Mouse Model

ObjectiveGlobally, there are approximately 15 million children who are born preterm each year. More clinical evidence proved that intrauterine infection or inflammation is the major risk factor for preterm birth, and is also the major cause of fetal brain injury and long-term adverse neurological outcomes. However, the pathogenesis of chorioamnionitis is concealed, and the clinical symptoms are not obvious. Its diagnosis mainly depends on the histological diagnosis. There are no effective interventions to the offspring injury. It is necessary to establish a model of intrauterine infection in pregnant mouse, and to research preterm birth outcome induced by chorioamnionitis, the purpose is to find and optimize an early intervention to improve preterm birth outcomes and protect the offspring brain injury.Recently, the studies find that N-acetyl-L-cysteine is also an antioxidant with anti-inflammatory properties and the regulation of apoptosis, can be used to reduce preterm birth and improve the neonatal outcome. People try to apply N-acetyl-L-cysteine to improve the outcomes of maternal and fetal induced by intrauterine inflammation. Another study suggests that activated microglia and astrocytes may be the potential therapeutic targets to deal with the fetal brain injury induced by intrauterine infection. Activated microglia having phagocytosis, is capable of uptake small as dendrimer nanoparticles. So administered targeted therapies will be the method to deal with the neuroinflammation. Based on the preliminary study, we employed maternally-administered DNAC in the established intrauterine infection pregnant mouse model to study:1) the influence of intrauterine infection on preterm birth and the offspring brain injury; 2) the preventive effect of DNAC on the preterm birth; 3) the protective effect of DNAC on the offspring brain injury; 4) the mechanisms of DNAC therapy on the preterm birth and the prenatal brain injury.MethodsTimed pregnant CD-1 mice were separated into non-surgery group and surgery group, surgery group was separated into NS and LPS groups. Timed pregnant CD-1 mice underwent laparotomy at embryonic day 17 (E17) and were injected with 25ug LPS in 100ul phosphate buffered saline (NS) or 100ul of NS vehicle between the first and second embryos of the right uterine horn. At 1 hour after the surgery was performed, dams from NS and LPS groups received 150ul of D-NAC (10 mg/kg).To explore:(1) Preterm birth was observed at 24 hours after the surgery was performed. (2) Behavior evaluation on postnatal day 5,9,13. (3) Harvested placenta and fetal brain at 6 hours after surgery was performed. Assay the expression of Caspase 3 activation in placenta and fetal brain; STAT3 and NFκB p65 activation and inflammatory response and CD3+T cell infiltration in placenta; embryonic microglia activation and postnatal microglia activation in fetal brain.Results1. DNAC reduced preterm birth rateThere was no difference between each group. Pregnant dams in the intrauterine LPS control group (n=12) had a preterm birth rate of 83%. Treatment of LPS-exposed dams with DNAC (n= 13) significantly reduced the preterm birth rate to 31%(p<0.05). In conclusion, the incidence of preterm birth reduced obviously by DNAC therapy.2. DNAC improved abnormal neurobehaviorIn order to evaluate the behavior, surface righting, cliff aversion, negative geotaxis, and ambulation neurobehavioral tests were administered on postnatal days 5, 9, and 13. Neuromotor development was evaluated by ambulation and negative geotaxis, cognitive by surface righting and cliff aversion. At PND5, no neonatal mice completed the ambulation test. LPS-exposed pups exhibited impaired performance compared to saline control; maternal administration of DNAC significantly improved test performance compared to LPS alone in other tests.3. DNAC reduced the activation of caspase 3 in fetal brainThe expression of caspase 3 in placenta and fetal brain was assessed by western blot. Caspase 3 expression and activation was not affected in the placenta at 6 hours. In the fetal brain, LPS induced the activation of caspase 3, and administration of DNAC significantly reduced activated caspase 3 compared to the LPS treated group.4. DNAC reduced acute inflammatory gene expression and inflammatory cytokine expression in placenta4.1 Both STAT3 and NFκB are important factors in the acute inflammation. The main subunit p65 of NFκB and the STAT3 should be phosphorylated when they are activated. We measured the expression of phosphorylated STAT3, total STAT3, the phosphorylated p65, and total p65 in the placenta. LPS induced increases in the ratios of pSTAT3/STAT3 compared to saline control, but not in p-p65/p65.4.2 We performed RT-qPCR to measure the expression of TNF-α, IL-1β, IL-10 and IL-6 in the placenta. In the placenta at 6 hours after surgery, expression of the inflammatory cytokines TNF-α, IL-1β, IL-10 and Il-6 were increased in the LPS-exposed group. Treatment with DNAC reduced the expression of these cytokines to control levels.5. DNAC suppressed CD3+ T-cell infiltration in the placentaAt 6 hours after LPS-exposure in the placenta, there was an increase in CD3+/CD45+ T-cells in both the maternal and fetal side of the placenta. Quantification of placental immune cells showed that DNAC treatment significantly reduced the LPS-induced infiltration of CD3+ T-cells.6. DNAC reduces LPS-induced microglia activation in offspring6.1 At 6 hours after LPS exposure in E17 embryos, fetal brains were analyzed by immunohistochemistry. The microglia marker Iba-1 was used to identify microglia in cortex and ventricular zone. We observed morphologic changes in microglia consistent with activation, including reduction of ramifications and adoption of amoeboid shape primarily in the ventricular zone6.2 At PND 17, neonatal brains were analyzed by immunohistochemistry and flow cytometry for microglia activation. We observed an increase in activated microglia morphology in LPS-exposed cortex and hippocampus compared to saline control, DNAC-treated, and nonsurgical control brainsFor flow cytometry analysis, LPS exposed offspring had a significant increase in activated microglia compared to saline control. DNAC treatment significantly reduced activated microglia in offspring compared to the LPS group.Conclusions1. DNAC might reduce preterm birth rate and improve the offspring neuromotor development and cognitive;2. The mechanic on DNAC therapy:1) DNAC might attenuate placental inflammation; maintain the maternal-fetal microenvironment;2) DNAC might reduce microglia activation in LPS-exposed offspring;3) DNAC might suppress the apoptosis of brain;4) DNAC might suppress peripheral immune cells infiltration in the placenta; speculate DNAC might play a role in protecting the placental barrier in early immune response.