Yafeng Dong

Brain Injury Caused by Chronic Fetal Hypoxemia Is Mediated by Inflammatory Cascade Activation

The prevalence of cerebral palsy (CP) shows little temporal or geographic variation and is associated with preterm birth, maternal/fetal infection/inflammation, and fetal growth restriction (IUGR), a potential surrogate for chronic fetal hypoxemia (CHX). We previously demonstrated CHX causes a fetal inflammatory response syndrome (FIRS). Herein, we test the hypothesis that CHX may cause fetal brain injury by upregulating inflammatory cytokine cascades, culminating in apoptosis pathway activation. Time-mated guinea pigs were housed in 12% or 10.5% O2 for the last 21% of gestation. Chronic fetal hypoxemia increased the lactate/pyruvate and decreased the glutathione (GSH)/oxidized glutathione (GSSH) ratios, confirming a shift to a prooxidant state. The end result was a >30% decrease in hippocampal neuron density. Based on a microarray spotted with 113 cytokines and receptors, 22 genes were upregulated by CHX in proportion to the degree of hypoxia; the findings were confirmed by quantitative polymerase chain reaction (PCR). Thus, CHX triggers fetal brain inflammation inversely proportional to its severity characterized by increased apoptosis and neuronal loss. We suggest CHX fetal brain injury is not directly caused by oxygen depravation but rather is an adaptive response that becomes maladaptive.

ATP-Binding Cassette Transporter Expression in Human Placenta as a Function of Pregnancy Condition

Fetal drug exposure is determined by the type and concentration of placental transporters, and their regulation is central to the development of new treatments and delivery strategies for pregnant women and their fetuses. We tested the expression of several clinically important transporters in the human placenta associated with various pregnancy conditions (i.e., labor, preeclampsia, and preterm labor-inflammation). Placentas were obtained from five groups of women at the time of primary cesarean section: 1) term no labor; 2) term labor; 3) preterm no labor (delivered for severe preeclampsia); 4) preterm labor without inflammation (PTLNI); and 5) preterm labor with inflammation (PTLI). Samples were analyzed by Western blot and immunohistochemistry to identify changes in protein expression. Relative mRNA expression was determined by quantitative real-time polymerase chain reaction. A functional genomic approach was used to identify placental gene expression and elucidate molecular events that underlie the given condition. Placental expression of ATP-binding cassette transporters from women in labor and women with preeclampsia was unaltered. Multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) and mRNA expression increased in placentas of women with preterm labor with inflammation. Molecular pathways of genes up-regulated in PTLI samples included cytokine-cytokine receptor interactions and inflammatory response compared with those in the PTLNI group. The mRNA expression of MDR1 and BCRP was correlated with that of interleukin-8, which also increased significantly in PTLI samples. These data suggest that the transfer of drugs across the placenta may be altered in preterm pregnancy conditions associated with inflammation through changes in MDR1 and BCRP.

Human Effector / Initiator Gene Sets That Regulate Myometrial Contractility During Term and Preterm Labor

OBJECTIVE Distinct processes govern transition from quiescence to activation during term (TL) and preterm labor (PTL). We sought gene sets that are responsible for TL and PTL, along with the effector genes that are necessary for labor independent of gestation and underlying trigger. STUDY DESIGN Expression was analyzed in term and preterm with or without labor (n=6 subjects/group). Gene sets were generated with logic operations. RESULTS Thirty-four genes were expressed similarly in PTL/TL but were absent from nonlabor samples (effector set); 49 genes were specific to PTL (preterm initiator set), and 174 genes were specific to TL (term initiator set). The gene ontogeny processes that comprise term initiator and effector sets were diverse, although inflammation was represented in 4 of the top 10; inflammation dominated the preterm initiator set. CONCLUSION TL and PTL differ dramatically in initiator profiles. Although inflammation is part of the term initiator and the effector sets, it is an overwhelming part of PTL that is associated with intraamniotic inflammation.

Chronic fetal hypoxia produces selective brain injury associated with altered nitric oxide synthases

OBJECTIVE The purpose of this study was to investigate the impact of chronic hypoxia on the nitric oxide synthase isoenzymes in specific brain structures. STUDY DESIGN Time-mated pregnant guinea pigs were exposed to 10.5% molecular oxygen for 14 days (animals with chronic fetal hypoxia; HPX) or room air (control animals; NMX); L-N6-(1-iminoethyl)-lysine (L-NIL; an inducible nitric oxide synthase inhibitor, 1 mg/kg/d) was administered to HPX group for 14 days (L-NIL + HPX). Fetal brains were harvested at term. Multilabeled immunofluorescence was used to generate a brain injury map. Laser capture microdissection and quantitative polymerase chain reaction were applied; cell injury markers, apoptosis activation, neuron loss, total nitric oxide, and the levels of individual nitric oxide synthase isoenzymes were quantified. RESULTS Chronic hypoxia causes selective fetal brain injury rather than global. Injury is associated with differentially affected nitric oxide synthases in both neurons and glial cells, with inducible macrophage-type nitric oxide synthase up-regulated at all injury sites. L-NIL attenuated the injury, despite continued hypoxia. CONCLUSION These studies demonstrate that chronic hypoxia selectively injures the fetal brain in part by the differential regulation of nitric oxide synthase isoenzymes in an anatomic- and cell-specific manner.

Chronic Hypoxemia Absent Bacterial Infection Is One Cause of the Fetal Inflammatory Response Syndrome (FIRS)

The object of the investigation was to determine whether chronic fetal hypoxemia triggers a systemic fetal inflammatory response absent bacterial infection. Chronically hypoxemic (10.5% O2) and lipopolysaccharide (LPS; 400 μg/kg of maternal body weight) injected intrauterine (but extra-amniotic) treated pregnant guinea pigs were used with appropriate controls. The presence of bacteria in the amniotic cavity was sought using polymerase chain reaction (PCR). Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) protein levels were measured in fetal sera and amniotic fluid (AF) by a commercially available, sensitive enzyme-linked immunosorbent assay (ELISA; IL-6 and TNF-α messenger RNA (mRNA) were also quantified in multiple fetal organs using real-time PCR. Prokaryotic DNA was not amplified from any sample, confirming the animals were not infected. Chronic hypoxemia dramatically increased IL-6 and TNF-α proteins in fetal sera and mRNA in lung, heart, and brain. There were no significant changes in either cytokine observed in the AF, fetal membranes, or fetal liver. Intrauterine but extra amniotic LPS also increased IL-6 and TNF-α protein in fetal sera and mRNA in lung, heart, and brain, plus increased the levels of both cytokines (protein/mRNA) in AF, fetal membranes, and fetal liver. Thus, an elevation in fetal blood IL-6 is not a specific marker of infection-induced fetal inflammatory response syndrome (FIRS). And in contrast to the fetal blood, an elevation in AF IL-6 seems associated only with LPS-induced FIRS.

Effect of Prostaglandin E2 on Multidrug Resistance Transporters In Human Placental Cells

Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades.

Chronic fetal hypoxia affects axonal maturation in guinea pigs during development: A longitudinal diffusion tensor imaging and T2 mapping study

To investigate the impact of chronic hypoxia on neonatal brains, and follow developmental alterations and adaptations noninvasively in a guinea pig model. Chronic hypoxemia is the prime cause of fetal brain injury and long‐term sequelae such as neurodevelopmental compromise, seizures, and cerebral palsy.

Prolactin Family of the Guinea Pig, Cavia porcellus

Prolactin (PRL) is a multifunctional hormone with prominent roles in regulating growth and reproduction. The guinea pig (Cavia porcellus) has been extensively used in endocrine and reproduction research. Thus far, the PRL cDNA and protein have not been isolated from the guinea pig. In the present study, we used information derived from the public guinea pig genome database as a tool for identifying guinea pig PRL and PRL-related proteins. Guinea pig PRL exhibits prominent nucleotide and amino acid sequence differences when compared with PRLs of other eutherian mammals. In contrast, guinea pig GH is highly conserved. Expression of PRL and GH in the guinea pig is prominent in the anterior pituitary, similar to known expression patterns of PRL and GH for other species. Two additional guinea pig cDNAs were identified and termed PRL-related proteins (PRLRP1, PRLRP2). They exhibited a more distant relationship to PRL and their expression was restricted to the placenta. Recombinant guinea pig PRL protein was generated and shown to be biologically active in the PRL-responsive Nb2 lymphoma cell bioassay. In contrast, recombinant guinea pig PRLRP1 protein did not exhibit PRL-like bioactivity. In summary, we have developed a new set of research tools for investigating the biology of the PRL family in an important animal model, the guinea pig.

Human ApoE ɛ2 Promotes Regulatory Mechanisms of Bioenergetic and Synaptic Function in Female Brain: A Focus on V-type H+-ATPase

Humans possess three major isoforms of the apolipoprotein E (ApoE) gene encoded by three alleles: ApoE ɛ2 (ApoE2), ApoE ɛ3 (ApoE3), and ApoE ɛ4 (ApoE4). It is established that the three ApoE isoforms confer differential susceptibility to Alzheimers disease (AD); however, an in-depth molecular understanding of the underlying mechanisms is currently unavailable. In this study, we examined the cortical proteome differences among the three ApoE isoforms using 6-month-old female, human ApoE2, ApoE3, and ApoE4 gene-targeted replacement mice and two-dimensional proteomic analyses. The results reveal that the three ApoE brains differ primarily in two areas: cellular bioenergetics and synaptic transmission. Of particular significance, we show for the first time that the three ApoE brains differentially express a key component of the catalytic domain of the V-type H+-ATPase (Atp6v), a proton pump that mediates the concentration of neurotransmitters into synaptic vesicles and thus is crucial in synaptic transmission. Specifically, our data demonstrate that ApoE2 brain exhibits significantly higher levels of the B subunit of Atp6v (Atp6v1B2) when compared to both ApoE3 and ApoE4 brains, with ApoE4 brain exhibiting the lowest expression. Our additional analyses show that Atp6v1B2 is significantly impacted by aging and AD pathology and the data suggest that Atp6v1B2 deficiency could be involved in the progressive loss of synaptic integrity during early development of AD. Collectively, our findings indicate that human ApoE isoforms differentially modulate regulatory mechanisms of bioenergetic and synaptic function in female brain. A more efficient and robust status in both areas-in which Atp6v may play a role-could serve as a potential mechanism contributing to the neuroprotective and cognition-favoring properties associated with the ApoE2 genotype.