Sex differences in neonatal outcomes following prenatal opioid exposure.

The impact of the opioid epidemic on pregnant people and children is a growing public health crisis. Understanding how opioids affect the developing brain during pregnancy and postnatally remains a critical area of investigation. Biological sex plays a crucial role in all physiologic processes, with the potential for a significant impact on neonatal outcomes, including those infants with opioid exposure. Here, we aim to explore current literature on the effect of sex on neonatal outcomes following prenatal opioid exposure. Sex differences in adults with opioid use disorder have been well studied, including increased mortality among males and higher rates of psychiatric comorbidities and likelihood of relapse in females. However, such differences are not yet well understood in neonates. Emerging clinical data suggest sex-specific effects in infants with prenatal opioid exposure on the expression of genes related to feeding regulation and reward signaling pathways. Increased susceptibility to white matter injury has also been noted in female infants following prenatal opioid exposure. Understanding the impact of sex as a biological variable on neonatal outcomes following prenatal opioid exposure is paramount to improving the health and well-being of infants, children, and adults impacted by the opioid epidemic.

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.

In utero methadone exposure permanently alters anatomical and functional connectivity: A preclinical evaluation.

The opioid epidemic is an ongoing public health crisis, and children born following prenatal opioid exposure (POE) have increased risk of long-term cognitive and behavioral sequelae. Clinical studies have identified reduced gray matter volume and abnormal white matter microstructure in children with POE but impacts on whole-brain functional brain connectivity (FC) have not been reported. To define effects of POE on whole brain FC and white matter injury in adult animals, we performed quantitative whole-brain structural and functional MRI. We used an established rat model of POE in which we have previously reported impaired executive function in adult rats analogous to persistent neurocognitive symptoms described in humans with POE. Pregnant Sprague-Dawley rat dams received continuous methadone (12 mg/kg/day) vs. saline infusion for 28 days via osmotic mini-pumps, exposing rats to pre- and postnatal opioid until weaning. At young adult age (P60), POE and saline exposed offspring underwent in vivo MRI included diffusion tensor imaging and functional MRI (fMRI). Results indicate that fractional anisotropy (FA) was decreased in adult animals with POE [n = 11] compared to animals that received saline [n = 9] in major white matter tracts, including the corpus callosum (p < 0.001) and external capsule (p < 0.01). This change in FA was concomitant with reduced axial diffusivity in the external capsule (p < 0.01) and increased radial diffusivity in the corpus callosum (p < 0.01). fMRI analyses reveal brainwide FC was diffusely lower in POE (p < 10-6; 10% of variance explained by group). Decreased connectivity in cortical-cortical and cortico-basal ganglia circuitry was particularly prominent with large effect sizes (Glass's Δ > 1). Taken together, these data confirm POE reduces brainwide functional connectivity as well as microstructural integrity of major white matter tracts. Altered neural circuitry, dysregulated network refinement, and diffuse network dysfunction have been implicated in executive function deficits that are common in children with POE. FC may serve as a translatable biomarker in children with POE.

Corrigendum: Methadone alters the peripheral inflammatory and central immune landscape following prenatal exposure in rats.

[This corrects the article DOI: 10.3389/adar.2022.10792.].

Methadone alters the peripheral inflammatory and central immune landscape following prenatal exposure in rats.

Opioid use during pregnancy continues to rise at alarming rates with a parallel trend in the number of infants and children exposed to opioid medications each year. Prenatal opioid exposure (POE) occurs at a critical timepoint in neurodevelopment disrupting intricate pathways essential for neural-immune maturation with the potential for devastating long-term consequences. Understanding the mechanisms underlying injury associated with POE is essential to address long-term outcomes and identify diagnostic and therapeutic biomarkers in this vulnerable patient population. Using an established preclinical model of POE, we investigated changes in cerebral and peripheral inflammation and peripheral blood mononuclear cell (PBMC) activity. We hypothesized that neuroinflammation, as defined by changes in specific cerebral immune cell populations, would exist in adult rats following POE concomitant with sustained peripheral immune hyperreactivity (SPIHR). Our data demonstrated alterations in cerebral immune cells at postnatal day 60 (P60) typified by increased regulatory T cells (p < 0.01) and neutrophils (p < 0.05) in rats with POE compared to controls. Evaluation of serum revealed increased levels of IL-6 (p < 0.05) and CXCL1 (p < 0.05) at P21 in rats with POE compared to controls with no significant difference in cytokine or chemokine levels between the two groups at P60. Additionally, PBMCs isolated from rats with POE at P21 demonstrated baseline hypersecretion of IL-6 (p < 0.01) and SPIHR with increased levels of TNF-α (p < 0.05) and CXCL1 (p < 0.05) following stimulation with LPS. At P60, however, there was no significant difference found in cytokine or chemokine levels secreted by PBMCs isolated from rats with POE at baseline or with LPS stimulation when compared to controls. Taken together, these data demonstrate cerebral inflammation months after prenatal opioid exposure and long after the resolution of systemic inflammation and SPIHR seen at toddler age equivalent. Chronic alterations in the cerebral immune cell populations secondary to prenatal opioid exposure may underly long-term consequences of developmental brain injury including deficits in cognition and attention. These findings may be invaluable to further investigations of precise biomarkers of injury and targeted therapeutics for this vulnerable population.

Sustained peripheral immune hyper-reactivity (SPIHR): an enduring biomarker of altered inflammatory responses in adult rats after perinatal brain injury.

BACKGROUND: Chorioamnionitis (CHORIO) is a principal risk factor for preterm birth and is the most common pathological abnormality found in the placentae of preterm infants. CHORIO has a multitude of effects on the maternal-placental-fetal axis including profound inflammation. Cumulatively, these changes trigger injury in the developing immune and central nervous systems, thereby increasing susceptibility to chronic sequelae later in life. Despite this and reports of neural-immune changes in children with cerebral palsy, the extent and chronicity of the peripheral immune and neuroinflammatory changes secondary to CHORIO has not been fully characterized. METHODS: We examined the persistence and time course of peripheral immune hyper-reactivity in an established and translational model of perinatal brain injury (PBI) secondary to CHORIO. Pregnant Sprague-Dawley rats underwent laparotomy on embryonic day 18 (E18, preterm equivalent). Uterine arteries were occluded for 60 min, followed by intra-amniotic injection of lipopolysaccharide (LPS). Serum and peripheral blood mononuclear cells (PBMCs) were collected at young adult (postnatal day P60) and middle-aged equivalents (P120). Serum and PBMCs secretome chemokines and cytokines were assayed using multiplex electrochemiluminescent immunoassay. Multiparameter flow cytometry was performed to interrogate immune cell populations. RESULTS: Serum levels of interleukin-1ß (IL-1ß), IL-5, IL-6, C-X-C Motif Chemokine Ligand 1 (CXCL1), tumor necrosis factor-α (TNF-α), and C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) were significantly higher in CHORIO animals compared to sham controls at P60. Notably, CHORIO PBMCs were primed. Specifically, they were hyper-reactive and secreted more inflammatory mediators both at baseline and when stimulated in vitro. While serum levels of cytokines normalized by P120, PBMCs remained primed, and hyper-reactive with a robust pro-inflammatory secretome concomitant with a persistent change in multiple T cell populations in CHORIO animals. CONCLUSIONS: The data indicate that an in utero inflammatory insult leads to neural-immune changes that persist through adulthood, thereby conferring vulnerability to brain and immune system injury throughout the lifespan. This unique molecular and cellular immune signature including sustained peripheral immune hyper-reactivity (SPIHR) and immune cell priming may be a viable biomarker of altered inflammatory responses following in utero insults and advances our understanding of the neuroinflammatory cascade that leads to perinatal brain injury and later neurodevelopmental disorders, including cerebral palsy.