Chorioamnionitis disrupts erythropoietin and melatonin homeostasis through the placental-fetal-brain axis during critical developmental periods

Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI.Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry.Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels.Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms

Complex pattern of interaction between in utero hypoxia-ischemia and intra-amniotic inflammation disrupts brain development and motor function

Background: Infants born preterm commonly suffer from a combination of hypoxia-ischemia (HI) and infectious perinatal inflammatory insults that lead to cerebral palsy, cognitive delay, behavioral issues and epilepsy. Using a novel rat model of combined late gestation HI and lipopolysaccharide (LPS)-induced inflammation, we tested our hypothesis that inflammation from HI and LPS differentially affects gliosis, white matter development and motor impairment during the first postnatal month. Methods: Pregnant rats underwent laparotomy on embryonic day 18 and transient systemic HI (TSHI) and/or intra-amniotic LPS injection. Shams received laparotomy and anesthesia only. Pups were born at term. Immunohistochemistry with stereological estimates was performed to assess regional glial loads, and western blots were performed for protein expression. Erythropoietin ligand and receptor levels were quantified using quantitative PCR. Digigait analysis detected gait deficits. Statistical analysis was performed with one-way analysis of variance and post-hoc Bonferonni correction. Results: Microglial and astroglial immunolabeling are elevated in TSHI + LPS fimbria at postnatal day 2 compared to sham (both P < 0.03). At postnatal day 15, myelin basic protein expression is reduced by 31% in TSHI + LPS pups compared to shams (P < 0.05). By postnatal day 28, white matter injury shifts from the acute injury pattern to a chronic injury pattern in TSHI pups only. Both myelin basic protein expression (P < 0.01) and the phosphoneurofilament/neurofilament ratio, a marker of axonal dysfunction, are reduced in postnatal day 28 TSHI pups (P < 0.001). Erythropoietin ligand to receptor ratios differ between brains exposed to TSHI and LPS. Gait analyses reveal that all groups (TSHI, LPS and TSHI + LPS) are ataxic with deficits in stride, paw placement, gait consistency and coordination (all P < 0.001). Conclusions: Prenatal TSHI and TSHI + LPS lead to different patterns of injury with respect to myelination, axon integrity and gait deficits. Dual injury leads to acute alterations in glial response and cellular inflammation, while TSHI alone causes more prominent chronic white matter and axonal injury. Both injuries cause significant gait deficits. Further study will contribute to stratification of injury mechanisms in preterm infants, and guide the use of promising therapeutic interventions

Evidence for Sexual Dimorphism in the Response to TLR3 Activation in the Developing Neonatal Mouse Brain: A Pilot Study

Toll-like receptor (TLR)3 activation during the neonatal period produces responses linked to the origins of neuropsychiatric disorders. Although there is sexual dimorphism in neuropsychiatric disorders, it is unknown if brain responses to TLR3 activation are sex-specific. We hypothesized that poly I:C in a post-natal day (P)8 model induces a sexually dimorphic inflammatory responses. C57BL6 mice received intraperitoneal injection of poly I:C (10 mg/kg) or vehicle [normal saline (NS)] at P8. Pups were killed at 6 or 14 h for caspase 3 and 8 activity assays, NFkB ELISA, IRF3, AP1, and GFAP western blotting and cytokines/chemokines gene expression real time qRT-PCR (4–6/group). A second group of pups were killed at 24 h (P9) or 7 days (P15) after poly I:C to assess astrocytic (GFAP) and microglia (Iba1) activation in the hippocampus, thalamus and cortex using immunohistochemistry, and gene and protein expression of cytokines/chemokines using real time RT-PCR and MSD, respectively (4–6/group). Non-parametric analysis was applied. Six hours after poly I:C, caspase-3 and -8 activities in cytosolic fractions were 1.6 and 2.8-fold higher in poly I:C-treated than in NS-treated female mice, respectively, while gene expressions of pro-inflammatory cytokines were upregulated in both sexes. After poly I:C, IRF3 nuclear translocation occurred earlier (6 h) in female mice and later (14 h) in male mice. At 14 h after poly I:C, only male mice also had increased nuclear NFκB levels (88%, p &lt; 0.001) and GFAP expression coinciding with persistent IL-6 and FAS gene upregulation (110 and 77%, respectively; p &lt; 0.001) and IL-10 gene downregulation (-42%, p &lt; 0.05). At 24 h after poly I:C, IL-1β, CXCL-10, TNF-α, and MCP-1 were similarly increased in both sexes but at 7 days after exposure, CXCL-10 and INFγ were increased and IL-10 was decreased only in female mice. Accordingly, microglial activation persisted at 7 days after poly I:C in the hippocampus, thalamus and cortex of female mice. This preliminary study suggests that TLR3 activation may produce in the developing neonatal mouse brain a sexually dimorphic response with early activation of caspase-dependent pathways in female mice, activation of inflammatory cascades in both sexes, which then persists in female mice. Further well-powered studies are essential to confirm these sex-specific findings

Prenatal Hypoxia–Ischemia Induces Abnormalities in CA3 Microstructure, Potassium Chloride Co-Transporter 2 Expression and Inhibitory Tone

Infants who suffer perinatal brain injury, including those with encephalopathy of prematurity, are prone to chronic neurological deficits, including epilepsy, cognitive impairment, and behavioral problems, such as anxiety, inattention, and poor social interaction. These deficits, especially in combination, pose the greatest hindrance to these children becoming independent adults. Cerebral function depends on adequate development of essential inhibitory neural circuits and the appropriate amount of excitation and inhibition at specific stages of maturation. Early neuronal synaptic responses to γ-amino butyric acid (GABA) are initially excitatory. During the early postnatal period, GABAAR responses switch to inhibitory with the upregulation of potassium-chloride co-transporter KCC2. With extrusion of chloride by KCC2, the Cl− reversal potential shifts and GABA and glycine responses become inhibitory. We hypothesized that prenatal hypoxic–ischemic brain injury chronically impairs the developmental upregulation of KCC2 that is essential for cerebral circuit formation. Following late gestation hypoxia–ischemia (HI), diffusion tensor imaging in juvenile rats shows poor microstructural integrity in the hippocampal CA3 subfield, with reduced fractional anisotropy and elevated radial diffusivity. The loss of microstructure correlates with early reduced KCC2 expression on NeuN-positive pyramidal neurons, and decreased monomeric and oligomeric KCC2 protein expression in the CA3 subfield. Together with decreased inhibitory post-synaptic currents during a critical window of development, we document for the first time that prenatal transient systemic HI in rats impairs hippocampal CA3 inhibitory tone. Failure of timely development of inhibitory tone likely contributes to a lower seizure threshold and impaired cognitive function in children who suffer perinatal brain injury

Embracing oligodendrocyte diversity in the context of perinatal injury

Emerging evidence is fueling a new appreciation of oligodendrocyte diversity that is overturning the traditional view that oligodendrocytes are a homogenous cell population. Oligodendrocytes of distinct origins, maturational stages, and regional locations may differ in their functional capacity or susceptibility to injury. One of the most unique qualities of the oligodendrocyte is its ability to produce myelin. Myelin abnormalities have been ascribed to a remarkable array of perinatal brain injuries, with concomitant oligodendrocyte dysregulation. Within this review, we discuss new insights into the diversity of the oligodendrocyte lineage and highlight their relevance in paradigms of perinatal brain injury. Future therapeutic development will be informed by comprehensive knowledge of oligodendrocyte pathophysiology that considers the particular facets of heterogeneity that this lineage exhibits

Embracing oligodendrocyte diversity in the context of perinatal injury.

Emerging evidence is fueling a new appreciation of oligodendrocyte diversity that is overturning the traditional view that oligodendrocytes are a homogenous cell population. Oligodendrocytes of distinct origins, maturational stages, and regional locations may differ in their functional capacity or susceptibility to injury. One of the most unique qualities of the oligodendrocyte is its ability to produce myelin. Myelin abnormalities have been ascribed to a remarkable array of perinatal brain injuries, with concomitant oligodendrocyte dysregulation. Within this review, we discuss new insights into the diversity of the oligodendrocyte lineage and highlight their relevance in paradigms of perinatal brain injury. Future therapeutic development will be informed by comprehensive knowledge of oligodendrocyte pathophysiology that considers the particular facets of heterogeneity that this lineage exhibits

Erythropoietin Modulates Cerebral and Serum Degradation Products from Excess Calpain Activation following Prenatal Hypoxia-Ischemia.

Preterm infants suffer central nervous system (CNS) injury from hypoxia-ischemia and inflammation - termed encephalopathy of prematurity. Mature CNS injury activates caspase and calpain proteases. Erythropoietin (EPO) limits apoptosis mediated by activated caspases, but its role in modulating calpain activation has not yet been investigated extensively following injury to the developing CNS. We hypothesized that excess calpain activation degrades developmentally regulated molecules essential for CNS circuit formation, myelination and axon integrity, including neuronal potassium-chloride co-transporter (KCC2), myelin basic protein (MBP) and phosphorylated neurofilament (pNF), respectively. Further, we predicted that post-injury EPO treatment could mitigate CNS calpain-mediated degradation. Using prenatal transient systemic hypoxia-ischemia (TSHI) in rats to mimic CNS injury from extreme preterm birth, and postnatal EPO treatment with a clinically relevant dosing regimen, we found sustained postnatal excess cortical calpain activation following prenatal TSHI, as shown by the cleavage of alpha II-spectrin (αII-spectrin) into 145-kDa αII-spectrin degradation products (αII-SDPs) and p35 into p25. Postnatal expression of the endogenous calpain inhibitor calpastatin was also reduced following prenatal TSHI. Calpain substrate expression following TSHI, including cortical KCC2, MBP and NF, was modulated by postnatal EPO treatment. Calpain activation was reflected in serum levels of αII-SDPs and KCC2 fragments, and notably, EPO treatment also modulated KCC2 fragment levels. Together, these data indicate that excess calpain activity contributes to the pathogenesis of encephalopathy of prematurity. Serum biomarkers of calpain activation may detect ongoing cerebral injury and responsiveness to EPO or similar neuroprotective strategies