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Perinatal hypoxia is a major cause of infant brain damage, lifelong neurological disability, and infant mortality. N-Acetyl-Cysteine (NAC) is a powerful antioxidant that acts directly as a scavenger of free radicals. We hypothesized that maternal-antenatal and offspring-postnatal NAC can protect offspring brains from hypoxic brain damage.Sixty six newborn rats were randomized into four study groups. Group 1: Control (CON) received no hypoxic intervention. Group 2: Hypoxia (HYP)-received hypoxia protocol. Group 3: Hypoxia-NAC (HYP-NAC). received hypoxia protocol and treated with NAC following each hypoxia episode. Group 4: NAC Hypoxia (NAC-HYP) treated with NAC during pregnancy, pups subject to hypoxia protocol. Each group was evaluated for: neurological function (Righting reflex), serum proinflammatory IL-6 protein levels (ELISA), brain protein levels: NF-κB p65, neuronal nitric oxide synthase (nNOS), TNF-α, and IL-6 (Western blot) and neuronal apoptosis (histology evaluation with TUNEL stain). Hypoxia significantly increased pups brain protein levels compared to controls. NAC administration to dams or offspring demonstrated lower brain NF-κB p65, nNOS, TNF-α and IL-6 protein levels compared to hypoxia alone. Hypoxia significantly increased brain apoptosis as evidenced by higher grade of brain TUNEL reaction. NAC administration to dams or offspring significantly reduce this effect. Hypoxia induced acute sensorimotor dysfunction. NAC treatment to dams significantly attenuated hypoxia-induced acute sensorimotor dysfunction. Prophylactic NAC treatment of dams during pregnancy confers long-term protection to offspring with hypoxia associated brain injury, measured by several pathways of injury and correlated markers with pathology and behavior. This implies we may consider prophylactic NAC treatment for patients at risk for hypoxia during labor.

Progesterone has been shown to regulate immunity during pregnancy, and progesterone administration may reduce inflammation-induced preterm labor. We sought to determine the maternal brain immune response to LPS-induced inflammation in pregnant and non-pregnant mice and whether additional progesterone supplementation attenuates this response. Pregnant (P: n = 9) and non-pregnant mice (NP: n = 9) were randomized to pretreatment with vaginal progesterone/carrier (Replens), daily from days 13 to 16. On days 15 and 16, LPS/saline was administered by intraperitoneal injection (Replens + saline n = 3; Replens + LPS n = 3; progesterone + LPS n = 3). Mice were sacrificed on day 16 and maternal serum analyzed for IL-6 levels and brains analyzed for nNOS, NF-kB, IL-6 protein levels and for immature myeloid cells (IMCs) and microglial activity. LPS significantly increased brain nNOS, NF-kB, and IL-6 in both NP and P mice, with significantly greater responses in P mice. In both NP and P groups, progesterone significantly attenuated LPS-induced increase of nNOS and NF-kB, however with no effect on serum IL-6. In the NP brains, LPS significantly increased IMC population and progesterone reduced the IMC phenotype to levels similar to controls. In P mice, neither LPS nor LPS + progesterone altered the brain IMC population. LPS significantly increased the microglial activity in both NP and P groups, which was attenuated by progesterone. Progesterone attenuates brain inflammatory response to LPS in both NP and P mice although it has no effect on systemic inflammation. In NP mice, progesterone attenuated the increase in brain IMC following LPS administration. Our results suggest that endogenous progesterone during pregnancy may protect the brain from LPS-induced inflammation.

MgSO 4 has been used for the past two decades as neuroprotective treatment in a variety of preterm conditions. Despite the putative advantages of MgSO 4 as a neuroprotective agent in the preterm brain, the short- and long-term molecular function of MgSO 4 as a neuroprotective agent has not been fully elucidated. Neuregulin (NRG1)-ErbB4 signaling plays a critical role in embryonic brain development, in the biology of dopaminergic, GABAergic, and glutamatergic systems. We hypothesize that this pathway may be associated with the neuroprotective role of MgSO 4 . The current study aims to investigate the ability of MgSO 4 to modulate the normal developing expression pattern of selected genes related to the NRG1-ErbB, dopaminergic, GABAergic, and glutamatergic systems. We demonstrate that overall short-term treatment of dam rats with MgSO 4 affects the expression of fetal brain NRG1, NRG3, ErbB4, GAD67, tyrosine hydroxylase (TH), dopamine D 2 and D 1 receptors, GluN1, and GluN2B. More specifically, the administration of MgSO 4 alters the expression of NRG-ErbB, GAD67, TH, and D2R at early gestation day 16 (GD16) regardless of the activation of the maternal immune system by lipopolysaccharide (LPS). Our data suggest that MgSO 4 treatment may affect the expression of major neuronal systems and pathways mostly at an early gestation day. These changes might be an initial clue (foundation stone) in the molecular mechanism that underlies the beneficial effect of MgSO 4 as a neuroprotective agent for the developmental brain.

Maternal natural vaginal progesterone (nVP) administration has been shown to reduce the risk of preterm birth (PTB). The largest randomized trial of nVP for PTB (OPPTIMUM) noted a sonographic reduction in neonatal brain injury following nVP treatment. We investigated the neuroinflammatory protective effect of maternal nVP in a mouse model for maternal inflammation. Pregnant mice (n = 24) were randomized to nVP (1 mg/day) or vehicle from days 13-16 of gestation. At days 15 and 16, lipopolysaccharide (30 μg) or saline were administered. Mice were sacrificed 4 h following the last injection. Fetal brains and placentas were collected. Levels of NF-κB, nNOS, IL-6, and TNFα were determined by Western blot. Maternal lipopolysaccharide significantly increased fetal brain levels of IL-6 (0.33 ± 0.02 vs. 0.11 ± 0.01 u), TNFα (0.3 ± 0.02 vs. 0.10 ± 0.01 u), NF-κB (0.32 ± 0.01 vs. 0.17 ± 0.01 u), and nNOS (0.24 ± 0.04 vs. 0.08 ± 0.01 u), and reduced the total glutathione levels (0.014 ± 0.001 vs. 0.026 ± 0.001 pmol/μl; p < 0.01) compared with control. Maternal nVP significantly reduced fetal brain levels of IL-6 (0.14 ± 0.01 vs. 0.33 ± 0.02 u), TNFα (0.2 ± 0.06 vs. 0.3 ± 0.02 u), NF-κB (0.16 ± 0.01 vs 0.32 ± 0.01 u), and nNOS (0.14 ± 0.01 vs 0.24 ± 0.04 u), and prevented the reduction of fetal brain total glutathione levels (0.022 ± 0.001 vs. 0.014 ± 0.001 pmol/μl; p < 0.01) to levels similar to controls. A similar pattern was demonstrated in the placenta. Maternal nVP for PTB may protect the fetal brain from inflammation-induced brain injury by inhibiting specific inflammatory and oxidative pathways in both brain and placenta.

Maternal infection/inflammation may induce fetal inflammatory responses, which have been associated with long-term offspring cerebral injury. We previously demonstrated that prophylactic N-Acetyl-Cysteine (NAC), administered prior to and following maternal lipopolysaccharide (LPS), reduced offspring cerebral injury as evident on MRI. In the present study, we used MRI to examine the effect of therapeutic NAC following maternal LPS-induced inflammation on neonatal brain injury. Pregnant Sprague-Dawley dams (n = 6) at day 18 of gestation received either intraperitoneal injection of LPS or saline (Control) at time 0. Animals were randomized to receive intravenous injection (tail vein) of NAC or saline at time +30 min. Pups were delivered spontaneously and allowed to mature until postnatal day 25. Male offspring (6-8 per group) were examined by MRI and analyzed using voxel-based analysis. Diffusion Tensor Imaging (DTI), an advanced MRI technique, was performed and quantitative parameters extracted (mean and radial diffusivity) and used to assess white and gray matter brain injury. Offspring of LPS-treated dams exhibited significantly increased mean, axial and radial diffusivity (RD) levels in white and gray matter consistent with cerebral injury. In contrast, offspring of NAC-treated LPS PS dams demonstrated reduced mean, axial and RD levels in most regions; similar to the saline group. Maternal NAC treatment following maternal inflammation significantly influenced brain micro-structure integrity as demonstrated by MRI-DTI scans. These findings suggest that maternal NAC therapy may be effective in human pregnancies associated with maternal/fetal inflammation, such as preterm rupture of membranes and chorioamnionitis., (Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Maternal inflammation is a risk factor for neonatal brain injury and future neurological deficits. Pomegranates have been shown to exhibit anti-inflammatory, anti-apoptotic and anti-oxidant activities., Objective: We hypothesized that pomegranate juice (POM) may attenuate fetal brain injury in a rat model of maternal inflammation., Study Design: Pregnant rats (24 total) were randomized for intraperitoneal lipopolysaccharide (100 μg/kg) or saline at time 0 at 18 days of gestation. From day 11 of gestation, 12 dams were provided ad libitum access to drinking water, and 12 dams were provided ad libitum access to drinking water with pomegranate juice (5 mL per day), resulting in 4 groups of 6 dams (saline/saline, pomegranate juice/saline, saline/lipopolysaccharide, pomegranate juice/lipopolysaccharide). All dams were sacrificed 4 hours following the injection and maternal blood and fetal brains were collected from the 4 treatment groups. Maternal interleukin-6 serum levels and fetal brain caspase 3 active form, nuclear factor-κB p65, neuronal nitric oxide synthase (phosphoneuronal nitric oxide synthase), and proinflammatory cytokine levels were determined by enzyme-linked immunosorbent assay and Western blot., Results: Maternal lipopolysaccharide significantly increased maternal serum interleukin-6 levels (6039 ± 1039 vs 66 ± 46 pg/mL; P < .05) and fetal brain caspase 3 active form, nuclear factor-κB p65, phosphoneuronal nitric oxide synthase, and the proinflammatory cytokines compared to the control group (caspase 3 active form 0.26 ± 0.01 vs 0.20 ± 0.01 U; nuclear factor-κB p65 0.24 ± 0.01 vs 0.1 ± 0.01 U; phosphoneuronal nitric oxide synthase 0.23 ± 0.01 vs 0.11 ± 0.01 U; interleukin-6 0.25 ± 0.01 vs 0.09 ± 0.01 U; tumor necrosis factor-α 0.26 ± 0.01 vs 0.12 ± 0.01 U; chemokine (C-C motif) ligand 2 0.23 ± 0.01 vs 0.1 ± 0.01 U). Maternal supplementation of pomegranate juice to lipopolysaccharide-exposed dams (pomegranate juice/lipopolysaccharide) significantly reduced maternal serum interleukin-6 levels (3059 ± 1121 pg/mL, fetal brain: caspase 3 active form (0.2 ± 0.01 U), nuclear factor-κB p65 (0.22 ± 0.01 U), phosphoneuronal nitric oxide synthase (0.19 ± 0.01 U) as well as the brain proinflammatory cytokines (interleukin-6, tumor necrosis factor-α and chemokine [C-C motif] ligand 2) compared to lipopolysaccharide group., Conclusion: Maternal pomegranate juice supplementation may attenuate maternal inflammation-induced fetal brain injury. Pomegranate juice neuroprotective effects might be secondary to the suppression of both the maternal inflammatory response and inhibition of fetal brain apoptosis, neuronal nitric oxide synthase, and nuclear factor-κB activation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Maternal magnesium administration has been shown to protect the preterm fetus from white- and gray-matter injury, although the mechanism is unknown., Objective: The purpose of the study is to test the following hypotheses: (1) maternal infections/inflammation activate fetal neuronal N-methyl-D-aspartate receptors that up-regulate neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells pathways; and (2) maternal magnesium sulfate attenuates fetal brain neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation through N-methyl-D-aspartate receptors., Study Design: Pregnant rats at embryonic day 16 and embryonic day 18 (n = 6, 48 total) received injections of intraperitoneal lipopolysaccharide 500 μg/kg or saline at time 0. Dams were randomized for treatment with subcutaneous magnesium sulfate (270 mg/kg) or saline for 2 hours prior to and following lipopolysaccharide/saline injections. At 4 hours after lipopolysaccharide administration, fetal brains were collected from the 4 treatment groups (lipopolysaccharide/saline, lipopolysaccharide/magnesium sulfate, saline/magnesium sulfate, saline/saline), and phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and chemokine (C-C motif) ligand 2 protein levels were determined by Western blot. An additional group of pregnant rats (n = 5) received N-methyl-D-aspartate-receptor antagonist following the lipopolysaccharide injection to study magnesium sulfate protective mechanism., Results: Lipopolysaccharide (lipopolysaccharide/saline) significantly increased fetal brain phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and chemokine (C-C motif) ligand 2 protein levels compared to the saline/saline group at both embryonic day 16 (phosphoneuronal nitric oxide synthase 0.23 ± 0.01 vs 0.11 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.24 ± 0.01 vs 0.14 ± 0.01 U; chemokine (C-C motif) ligand 2 0.28 ± 0.01 vs .01 ± 0.01 U) and embryonic day 18 (phosphoneuronal nitric oxide synthase 0.28 ± 0.01 vs 0.12 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.12 ± 0.01 vs 0.1 ± 0.01 U; chemokine (C-C motif) ligand 2 0.27 ± 0.01 vs 0.11 ± 0.01 U). Magnesium sulfate treatment to lipopolysaccharide dams (lipopolysaccharide/magnesium sulfate) significantly decreased fetal brain phosphoneuronal nitric oxide synthase, nuclear factor kappa-light-chain-enhancer of activated B cells, and chemokine (C-C motif) ligand 2 protein levels compared to lipopolysaccharide/saline dams at both embryonic day 16 (neuronal nitric oxide synthase 0.17 ± 0.02 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.17 ± 0.03 U; chemokine (C-C motif) ligand 2 0.18 ± 0.01 U) and embryonic day 18 (phosphoneuronal nitric oxide synthase 0.1 ± 0.01 U; nuclear factor kappa-light-chain-enhancer of activated B cells 0.09 ± 0.01 U; chemokine (C-C motif) ligand 2 0.21 ± 0.01 U). Notably, maternal lipopolysaccharide at embryonic day 16 activated nuclear factor kappa-light-chain-enhancer of activated B cells twice as often compared to dams induced at embryonic day 18. N-methyl-D-aspartate-receptor antagonist decreased fetal brain phosphoneuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells levels comparable to magnesium sulfate., Conclusion: Lipopolysaccharide-simulated inflammation during pregnancy may cause brain injury through activation of neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells pathways and, potentially, production of excitotoxic nitric oxide and inflammatory mediators. The increased susceptibility to brain injury in preterm fetuses may be due to enhanced nuclear factor kappa-light-chain-enhancer of activated B cells activation. Magnesium sulfate protective effects may be secondary, in part, to inhibition of neuronal nitric oxide synthase and nuclear factor kappa-light-chain-enhancer of activated B cells activation and decrease proinflammatory cytokine production through blocking nuclear factor kappa-light-chain-enhancer of activated B cells receptors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Maternal infection or inflammation may induce fetal inflammatory responses associated with fetal injury and cerebral palsy. We sought to assess the inflammation-associated neuroprotective potential of prophylactic N-acetyl-cysteine (NAC). We examined the effect of NAC on prevention of maternal lipopolysaccharide (LPS)-induced neonatal brain injury using magnetic resonance imaging., Study Design: Pregnant Sprague Dawley dams (n = 5-8) at embryonic day 18 received intraperitoneal injection of LPS or saline at time 0. Animals were randomized to receive 2 intravenous injections of NAC or saline (time -30 and 120 minutes). Pups were delivered spontaneously and allowed to mature until postnatal day 25. Female offspring were examined by magnetic resonance brain imaging and analyzed using voxel-based analysis after spatial normalization. T2 relaxation time was used to assess white matter injury and diffusion tensor imaging for apparent diffusion coefficient (ADC) to assess white and gray matter injury., Results: Offspring of LPS-treated dams exhibited significantly increased T2 levels and increased ADC levels in white and gray matter (eg, hypothalamus, motor cortex, corpus callosum, thalamus, hippocampus), consistent with diffuse cerebral injury. In contrast, offspring of NAC-treated LPS dams demonstrated similar T2 and ADC levels as control in both white and gray matter., Conclusion: Maternal NAC treatment significantly reduced evidence of neonatal brain injury associated with maternal LPS. These studies suggest that maternal NAC therapy may be effective in human deliveries associated with maternal/fetal inflammation., (Copyright © 2013 Mosby, Inc. All rights reserved.)